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Dengue-2-virus-interacting polypeptides involved in mosquito cell infection
Abstract
For the design of effective antiviral strategies, understanding the fundamental steps of the virus life cycle, including virus-host interactions, is essential. We performed a virus overlay protein binding assay followed by proteomics for identification of proteins from membrane fractions of A7 (Aedes aegypti) cells, C6/36 (Aedes albopictus) cells and the midgut brush border membrane fraction of Ae. aegypti mosquito that bind to dengue-2 virus. Actin, ATP synthase β subunit, HSc 70, orisis, prohibitin, tubulin β chain, and vav-1 were identified as dengue-2-virus-binding proteins. Our results suggest that dengue-2 virus exploits an array of housekeeping proteins for its entry in mosquito cells.
... [50] [51] [52] ...
... Apart from the studies focusing on proteomic changes upon DENV infection, the virus overlay protein binding assay (VOPBA) has been used to identify cellular receptors for DENV [48][49][50][51]. This approach has identified enolase, cadherin, beta-adrenergic receptor kinase (beta-ARK) and translation elongation factor EF-1 alpha/Tu as potential DENV binding receptors in Ae. aegypti midgut homogenates and C6/36 cell lysates [50]. ...
... This approach has identified enolase, cadherin, beta-adrenergic receptor kinase (beta-ARK) and translation elongation factor EF-1 alpha/Tu as potential DENV binding receptors in Ae. aegypti midgut homogenates and C6/36 cell lysates [50]. Other proteins such as actin, orisis, vav-1, prohibitin, ATP synthase β subunit, tubulin β chain and 70 kD heat shock cognate protein (HSC70) were also shown to bind to the membrane fractions of Ae. aegypti-derived A7 cells and C6/36 cells, and the midgut brush border membrane fraction of Ae. aegypti [51]. Independently, another group has also identified HSC70 interacting with DENV in C6/36 cells, together with 78 kDa glucose-regulated protein (GRP78 or BiP), 70 kDa heat shock protein (HSP70) and 40 kDa protein with homology to protein disulfide isomerase (PDI) [52]. ...
Dengue virus (DENV), like other viruses, closely interacts with the host cell machinery to complete its life cycle. Over the course of infection, DENV interacts with several host factors with pro-viral activities to support its infection. Meanwhile, it has to evade or counteract host factors with anti-viral activities which inhibit its infection. These molecular virus-host interactions play a crucial role in determining the success of DENV infection. Deciphering such interactions is thus paramount to understanding viral fitness in its natural hosts. While DENV-mammalian host interactions have been extensively studied, not much has been done to characterize DENV-mosquito host interactions despite its importance in controlling DENV transmission. Here, to provide a snapshot of our current understanding of DENV-mosquito interactions, we review the literature that identified host factors and cellular processes related to DENV infection in its mosquito vectors, Aedes aegypti and Aedes albopictus , with a particular focus on DENV-mosquito omics studies. This knowledge provides fundamental insights into the DENV life cycle, and could contribute to the development of novel antiviral strategies.
... Isolation of the midgut brush border membrane fraction (BBMF) from Ae. aegypti: BBMFs from the midgut epithelial cells of Ae. aegypti larvae and adults were prepared as described by Mourya et al15 and Paingankar et al16. ...
... A recent report by Wintachai et al23 also demonstrated HSP60 as a CHIKV interacting protein and have claimed it as a potential candidate responsible for mediating the entry of CHIKV into the host cell. Earlier studies demonstrated prohibitin, Ae. aegypti midgut surface protein as DENV2 interacting protein162325. In the current study, interaction of prohibitin with CHIKV was not observed. ...
... However, Alphavirus receptor/s have not been conclusively identified15. Arboviruses, including Japanese encephalitis22 chikungunya23 and DENV1624 have been reported to use HSP family proteins as their cell receptors. In the VOPBA assay, we detected a 60 KDa heat shock protein as a CHIKV interacting protein and potential receptor for the entry into mosquito midgut cells. ...
Background & objectives:
The susceptibility of the mosquito to the invading pathogen is predominantly dictated by the complex interactions between the mosquito midgut and the surface proteins of the invading pathogen. It is well documented that the midgut microbiota plays an important role in determining the susceptibility of the mosquito to the pathogen. In the present study, we investigated the influence of Serratia odorifera, an endogenous cultivable midgut inhabitant of Aedes aegypti on the chikungunya virus (CHIKV) susceptibility to this mosquito.
Methods:
Ae. aegypti females free of gutflora were co-fed with CHIKV and either of the two midgut inhabitants namely, S. odorifeara and Microbacterium oxydans. CHIKV dissemination was checked on 10th day post feeding (DPF) using indirect immunoflurescence assay and plaque assay. CHIKV interacting proteins of the mosquito midgut were identified using virus overlay protein binding assay and MALDI TOF/TOF analysis.
Results:
The observations revealed that co-feeding of S. odorifera with CHIKV significantly enhanced the CHIKV susceptibility in adult Ae. aegypti, as compared to the mosquitoes fed with CHIKV alone and CHIKV co-fed with another midgut inhabitant, M. oxydans. Virus overlay protein binding assay (VOPBA) results revealed that porin and heat shock protein (HSP60) of Ae. aegypti midgut brush border membrane fraction interacted with CHIKV.
Interpretation & conclusions:
The results of this study indicated that the enhancement in the CHIKV susceptibility of Ae. aegypti females was due to the suppression of immune response of Ae. aegypti as a result of the interaction between S. odorifera P40 protein and porin on the gut membrane.
... Membrane fractions were isolated as described earlier by Paingankar et al. (2010) and Apte-deshpande et al. (2012). Cells were washed with buffer A (0.3 M mannitol, 5 mM EGTA, 20 mM Tris/HCl, 2 mM PMSF, pH 7.4) and homogenized in buffer B (0.3 M mannitol, 5 mM EGTA, 20 mM Tris/HCl, 2 mM PMSF, Triton X-100, pH 7.4). ...
... This study reports binding of actin, STAT-2 and HSP70 proteins with CHIKV in two mammalian cell lines based on VOPBA and subsequent protein identification. The results of earlier studies suggested that while proteins may be denatured as part of VOPBA, the technique is still capable of selecting physiologically relevant binding molecules, possibly as a result of partial renaturation of proteins during the overlay process (Upanan et al. 2008;Paingankar et al. 2010;Apte-deshpande et al. 2012). We used kidney cell lines of human and monkey origin and three CHIKV strains for the identification of virus binding proteins. ...
... However, the alphavirus receptor/s has not been conclusively identified. Animal viruses, including human T lymphotropic virus type 1 (HTLV-1) (Sagara et al. 1998), rotavirus (Guerrero et al. 2002), Japanese encephalitis (Zhu et al. 2012) and dengue virus (Reyes-Del Valle et al. 2005;Paingankar et al. 2010) have been reported to use HSP70 family proteins as cell receptors. In the VOPBA assay, we detected HSP 70 as a CHIKV interacting protein. ...
Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.
... These gene/protein modifications within the host may represent the way arboviruses' take advantage of the mosquito's cytoskeleton by rearranging it into tracks to actively transport endosomes containing viral particles (Walsh and Naghavi, 2019). To facilitate transport at a later phase, direct interaction between arboviral proteins and host cytoskeletal motor proteins (e.g., dynein and myosin) occur (Paingankar et al., 2010;Mairiang et al., 2013;Foo and Chee, 2015; FIGURE 1 | Ae. aegypti's cellular mechanisms are affected by arboviral infection. Arboviral infections alter Ae. aegypti's genes, proteins, and metabolites to control the host's cellular mechanisms. ...
... In the case of DENV infection in Ae. aegypti, link between non-structural (NS) protein 5 and myosin has been reported (Mairiang et al., 2013). Similarly, other cytoskeletal structures like actin and tubulin is said to interact with DENV to facilitate infection in vitro (Paingankar et al., 2010). Protein interaction network prediction in Ae. aegypti also suggests that tubulin is highly associated with DENV infection in the mosquito host with roles in transport and assembly (Guo et al., 2010). ...
Aedes aegypti is inherently susceptible to arboviruses. The geographical expansion of this vector host species has led to the persistence of Dengue, Zika, and Chikungunya human infections. These viruses take advantage of the mosquito’s cell to create an environment conducive for their growth. Arboviral infection triggers transcriptomic and protein dysregulation in Ae. aegypti and in effect, host antiviral mechanisms are compromised. Currently, there are no existing vaccines able to protect human hosts from these infections and thus, vector control strategies such as Wolbachia mass release program is regarded as a viable option. Considerable evidence demonstrates how the presence of Wolbachia interferes with arboviruses by decreasing host cytoskeletal proteins and lipids essential for arboviral infection. Also, Wolbachia strengthens host immunity, cellular regeneration and causes the expression of microRNAs which could potentially be involved in virus inhibition. However, variation in the magnitude of Wolbachia’s pathogen blocking effect that is not due to the endosymbiont’s density has been recently reported. Furthermore, the cellular mechanisms involved in this phenotype differs depending on Wolbachia strain and host species. This prompts the need to explore the cellular interactions between Ae. aegypti-arboviruses-Wolbachia and how different Wolbachia strains overall affect the mosquito’s cell. Understanding what happens at the cellular and molecular level will provide evidence on the sustainability of Wolbachia vector control.
... ATPsyn-β has also been shown to act as a receptor for viruses. For example, ATPsyn-β of mosquito cells act as a receptor to mediate the entry of chikungunya virus (CHIKV) and dengue-2 virus (Paingankar et al., 2010;Fongsaran et al., 2014). ...
... ATPsyn-β is typically involved in ATP synthesis and several other biological activities in cells (Moser et al., 1999(Moser et al., , 2001Martinez et al., 2003;Lin et al., 2009), and it also serves as a receptor for viruses (Paingankar et al., 2010;Fongsaran et al., 2014). Shrimp ATPsyn-β has been speculated to be a candidate receptor for WSSV. ...
White spot syndrome virus (WSSV) is highly virulent toward shrimp, and F1 ATP synthase β subunit (ATPsyn-β) has been suggested to be involved in WSSV infection. Therefore, in this study, interactions between Penaeus monodon ATPsyn-β (PmATPsyn-β) and WSSV structural proteins were characterized. Based on the results of yeast two-hybrid, co-immunoprecipitation, and protein pull-down assays, WSSV VP51B and VP150 were identified as being able to interact with PmATPsyn-β. Membrane topology assay results indicated that VP51B and VP150 are envelope proteins with large portions exposed outside the WSSV virion. Cellular localization assay results demonstrated that VP51B and VP150 co-localize with PmATPsyn-β on the membranes of transfected cells. Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA results demonstrated that VP51B and VP150 bound to PmATPsyn-β in a dose-dependent manner, which could be competitively inhibited by the addition of WSSV virions. In vivo neutralization assay results further showed that both recombinant VP51B and VP150 could delay mortality in shrimp challenged with WSSV.
... The 'ectopic' distribution of β-ATPase allows its functioning as a receptor of several ligands as well as its participation in different physiological and pathological processes. They include the binding of the high-density lipoprotein in the reverse cholesterol transport (Vantourout et al. 2010), the binding of juvenile hormone binding protein in insect endocrine regulation (Zalewska et al. 2009), and the binding of dengue-2 virus during mosquito cell infection (Paingankar et al. 2010). ...
... Each portion is formed by different subunits, being the α-and β-subunits part of the catalytic F 1 portion (Meier et al. 2011). At present, there are reports demonstrating that the β-ATPase in the plasma membrane not only serves as receptor of several ligands but also participates in different cellular and metabolic processes (Zalewska et al. 2009, Paingankar et al. 2010, Vantourout et al. 2010). In P. megistus, the occurrence of β-ATPase in the microsomal fraction strongly suggested its presence in the plasma membranes of fat body cells. ...
In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas’ disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the β-chain of ATP synthase (β-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the β-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in β-ATPase expression were found in fasting and fed insects. Blocking the lipophorin–β-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that β-ATPase functions as a lipophorin docking receptor in the fat body.
... Mosquito midgut BBMF proteins were isolated from adult female mosquito midgut epithelial cells as reported previously (Paingankar et al., 2010). Midguts were dissected from adult female mosquitoes (five mosquitoes per species) under a binocular microscope and the peritrophic membranes and malpighian tubules were removed. ...
... The presence of specific midgut epithelial receptors has been attributed, at least in part, to the difference in vector competence for virus transmission between mosquito species (Mercado-Curiel et al., 2008). It has been documented that most of the well characterized arbovirus receptors are housekeeping molecules that are also present ubiquitously in the midgut brush border membrane of mosquitoes (Paingankar et al., 2010). ...
... Mosquito midgut BBMF proteins were isolated from adult female mosquito midgut epithelial cells as reported previously (Paingankar et al., 2010). Midguts were dissected from adult female mosquitoes (five mosquitoes per species) under a binocular microscope and the peritrophic membranes and malpighian tubules were removed. ...
... The presence of specific midgut epithelial receptors has been attributed, at least in part, to the difference in vector competence for virus transmission between mosquito species (Mercado-Curiel et al., 2008). It has been documented that most of the well characterized arbovirus receptors are housekeeping molecules that are also present ubiquitously in the midgut brush border membrane of mosquitoes (Paingankar et al., 2010). ...
Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp. mosquitoes. The present study investigated vector competence for CHIKV in Aedes aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV DRDE-06. Infection of midguts and dissemination to heads was confirmed by immunofluorescence assay at different time points. A plaque assay was performed from mosquito homogenates at different time points to study CHIKV replication. Presence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was detected by virus overlay protein binding assay (VOPBA). The identity of these proteins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae. albopictus midguts and dissemination to heads was observed to be similar. A plaque assay performed with infected mosquito homogenates revealed that CHIKV replication dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA performed with mosquito midgut membrane proteins revealed that prohibitin could serve as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the non-permissiveness of these mosquitoes to infection.
... Protein binding assays and mass spectrometry analysis have identified several additional potential flavivirus cellular receptors (65)(66)(67). Among them, the tubulin and tubulin-like proteins in C6/36 Ae. albopictus cell line (65). ...
... The 70 kDa protein, also known as heat shock cognate protein (HSC70) or HSPA8, acts as a chaperone protein during DENV entry (70,71). Modulation of HSC70 expression was observed during DENV-2 infection, with an increase on the cell membrane during infection, suggesting that DENV-2 utilizes HSC70 for entry into mosquito cells (67). In addition to its role in viral entry, HSP70 is involved in virion biogenesis and RNA replication (71). ...
Flaviviruses are emerging and re-emerging arthropod-borne pathogens responsible for significant mortality and morbidity worldwide. The genus comprises more than seventy small, positive-sense, single-stranded RNA viruses, which are responsible for a spectrum of human and animal diseases ranging in symptoms from mild, influenza-like infection to fatal encephalitis and haemorrhagic fever. Despite genomic and structural similarities across the genus, infections by different flaviviruses result in disparate clinical presentations. This review focusses on two haemorrhagic flaviviruses, dengue virus and yellow fever virus, and two neurotropic flaviviruses, Japanese encephalitis virus and Zika virus. We review current knowledge on host-pathogen interactions, virus entry strategies and tropism.
... Aedes aegypti populations from India showed high vector susceptibility for CHIKV. It has been well documented that difference in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors 30 . Most of the well characterized arbovirus receptors are house keeping molecules and are also present ubiquitously in the midgut brush border membrane of mosquitoes 30 . ...
... It has been well documented that difference in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors 30 . Most of the well characterized arbovirus receptors are house keeping molecules and are also present ubiquitously in the midgut brush border membrane of mosquitoes 30 . Therefore, we hypothesized that at 100 per cent infection rate mosquito will show similar growth kinetics. ...
Background & objectives: Although having immense clinical relevance, yet only a few studies have been targeted to understand the chikungunya virus (CHIKV) susceptibility and growth in Aedes aegypti populations from India. This study was undertaken to investigate CHIKV susceptibility and growth kinetics in Ae. aegypti along with genetic heterogeneity of Ae. aegypti populations.
... Aedes aegypti populations from India showed high vector susceptibility for CHIKV. It has been well documented that difference in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors 30 . Most of the well characterized arbovirus receptors are house keeping molecules and are also present ubiquitously in the midgut brush border membrane of mosquitoes 30 . ...
... It has been well documented that difference in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors 30 . Most of the well characterized arbovirus receptors are house keeping molecules and are also present ubiquitously in the midgut brush border membrane of mosquitoes 30 . Therefore, we hypothesized that at 100 per cent infection rate mosquito will show similar growth kinetics. ...
Background & objectives: Although having immense clinical relevance, yet only a few studies have been targeted to understand the chikungunya virus (CHIKV) susceptibility and growth in Aedes aegypti populations from India. This study was undertaken to investigate CHIKV susceptibility and growth kinetics in Ae. aegypti along with genetic heterogeneity of Ae. aegypti populations.
Methods: Dose dependent CHIKV susceptibility and growth kinetic studies for three CHIKV strains reported from India were carried out in Ae. aegypti mosquito populations. The phenotypic variation and genetic heterogeneity in five Ae. aegypti populations were investigated using multivariate morphometrics and allozyme variation studies.
Results: The dissemination and growth kinetics studies of the three CHIKV strains showed no selective advantage for a particular strain of CHIKV in Ae. aegypti. At 100 per cent infection rate, five geographic Ae. aegypti populations showed differences in dissemination to three CHIKV strains. Morphometric studies revealed phenotypic variation in all the studied populations. The allelic frequencies, F statistics, and Nei′s genetic identity values showed that genetic differences between the populations were small, but significant.
Interpretation & conclusions: The results obtained in this study suggest that genetic background of the vector strongly influences the CHIKV susceptibility in Ae. aegypti.
... Aedes aegypti populations from India showed high vector susceptibility for CHIKV. It has been well documented that difference in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors 30 . Most of the well characterized arbovirus receptors are house keeping molecules and are also present ubiquitously in the midgut brush border membrane of mosquitoes 30 . ...
... It has been well documented that difference in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors 30 . Most of the well characterized arbovirus receptors are house keeping molecules and are also present ubiquitously in the midgut brush border membrane of mosquitoes 30 . Therefore, we hypothesized that at 100 per cent infection rate mosquito will show similar growth kinetics. ...
Abstract
Ingwavuma virus (INGV), a bunyavirus has been isolated repeatedly from birds, pigs and mosquitoes in India, South Africa and Southeast Asian countries. The virus is maintained in nature in a pig-mosquito-bird cycle closely similar to Japanese encephalitis virus epidemiology. Though human infection is rarely recorded, neutralizing antibodies in human serum have been detected in India and Africa. Hence, a study was designed to determine the potential of some of the locally prevalent mosquito species in replication and transmission of the virus. In the present study, INGV replication was seen in Culex quinquefasciatus and Cx tritaeniorhynchus mosquitoes with peak titers of 3.7 and 4.7 log10TCID50/ml respectively. Aedes aegypti mosquitoes maintained INGV till 16th day post infection (PI), but no evident multiplication was seen. Vector competence studies have shown the presence of INGV in the saliva of Cx quinquefasciatus mosquitoes from 8th day PI onwards while INGV could not be detected in saliva of Ae aegypti and Cx tritaeniorhynchus mosquitoes upto 20days PI. Horizontal transmission to infant mice was demonstrated by Cx quinquefasciatus mosquitoes but not by Cx tritaeniorhynchus. However,vertical transmission could not be demonstrated in any of the mosquitoes. Though no major outbreaks involving humans has been reported yet, the potential of the virus to replicate in different species of mosquitoes and vertebrates including humans makes it a pathogen of public health importance.
... Hsp-related protein 45 kD 4 [55] Tubulin-like protein 48 kD 2 [56] Laminin receptor 3, 4 [54] C6/36, CCl-125 (A. aegypti), adult A. aegypti Prohibitin 2 [57,58] AP-61 (A. pseudoculterallis) Glycosphingolipid 2 [45] C6/36, midgut A. aegypti Unknown protein 67-80 kD 1-4 [52] Salivary glands A. aegypti and A. polynesiensis Unknown protein 48-77 kD 1-4 [53] Salivary glands, midgut, ovary A. aegypti Unknown protein 45 kD 4 [51] DC-SIGN: Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin; GRP-78: Glucose-regulated protein 78; Hsp: Heat-shock protein; TAM: Tyro3, Axl and Mer; TIM: T-cell immunoglobulin and mucin domain containing proteins. Macrophages are also major targets of human infection with DENV and another lectin, the mannose receptor, was identified as a DENV receptor for the four DENV serotypes through binding and functional analyses [33]. ...
... More recent future science group www.futuremedicine.com investigations have identified more accurately some cellular proteins with DENV-binding ability in mosquito cells, including a laminin-binding protein, a Hsp90-related protein, prohibitin and a tubulin-like protein [54][55][56][57][58]. Finally, as also shown in mammalian cells, glycosphingolipids specific for mosquito cells were found reactive with DENV-2 [45]. ...
Dengue virus (DENV) is the etiological agent of the most important human viral infection transmitted by mosquitoes in the world. In spite of the serious health threat that dengue represents, at present there are no vaccine or antiviral agents available and treatment of patients consists of supportive therapy. This review will focus on the process of DENV entry into the host cell as a potential target for antiviral therapy. The recent advances in the knowledge of viral and cellular molecules and mechanisms involved in binding, internalization and trafficking of DENV into the host cell until virion uncoating are discussed, together with an overview of the strategies and compounds evaluated for development of antiviral agents targeted to DENV entry.
... There have been few reports of the involvement of PHB in DENV infection. One report identified PHB as a DENV receptor protein expressed on the surface of insect cells 27 , and other studies have supported the possible involvement of PHB in infection of insect cells by identifying PHB as a DENV E protein interacting protein expressed on the cell surface 49 . One further study has identified prohibitin as a receptor molecule mediating entry of DENV 3 into human neuroblastoma (SH-SY5Y) and microglial (CHME-3) cells 50 . ...
The mosquito transmitted dengue virus (DENV) is a major public health problem in many tropical and sub-tropical countries around the world. Both vaccine development and drug development are complex as the species Dengue virus consist of four distinct viruses (DENV 1 to DENV 4) each of which is composed of multiple lineages and strains. To understand the interaction of DENV with the host cell machinery, several studies have undertaken in vitro proteomic analysis of different cell lines infected with DENV. Invariably, these studies have utilized DENV 2. In this study we sought to define proteins that are differentially regulated by two different DENVs, DENV 2 and DENV 4. A 2-dimensional proteomic analysis identified some 300 protein spots, of which only 11 showed differential expression by both DENVs. Of these, only six were coordinately regulated. One protein, prohibitin 1 (PHB1) was downregulated by infection with both DENVs. Overexpression of PHB1 increased DENV protein expression, level of infection and genome copy number. DENV E protein colocalized with PHB, and there was a direct interaction between DENV 2 E protein and PHB1, but not between DENV 4 E protein and PHB1. The low number of proteins showing coordinate regulation after infection by different DENVs is a cause for concern, particularly in determining new druggable targets, and suggests that studies should routinely investigate multiple DENVs.
... Effective DENV infection in mosquito cells is essentially achieved by the cooperation of several proteins (Guo et al., 2010). Actin and tubulin support DENV infection in vitro, while NS5 associates with myosin in DENV infection (Paingankar et al., 2010). Tubulin is expected to be an important component in the transit and assembly of DENV in mosquitoes. ...
Dengue virus (DENV) is spread by Aedes aegypti and causes significant global health risks. Alternative techniques are urgently needed because the current control mech-anisms are insufficient to reduce the transmission of DENV. Introducing Wolbachia pipientis into Ae. aegypti inhibits DENV transmission, however, the underlying mechanisms are still poorly understood. Innate immune effector upregulation, the regulation of autophagy, and intracellular competition between Wolbachia and DENV for lipids are among the theories for the mechanism of inhibition. Further-more, mainly three immune pathways Toll, IMD and JAK/STAT are involved in the host for the suppression of the virus. These pathways are activated by Wolbachia and DENV in the host and are responsible for the upregulation and downregulation of many genes in mosquitoes, which ultimately reduces the titer of the DENV in the host. The functioning of these immune pathways depends upon the Wolbachia, host and virus interaction. Here, we summarize the current understanding of DENV recognition by the Ae. aegypti’s immune system, aiming to create a comprehensive picture of our knowledge. Additionally, we investigated how Wolbachia regulates ac-tivation of multiple genes associated with the Toll and IMD pathway, for the reduc-tion of arboviruses.
... Increased Vav levels and Vav phosphorylation are also associated with Vav2 binding of the L2 protein of bovine papilloma virus and have been proposed to affect viral entry and oncogenic potential (32). In relation to DENV, in a screen to identify cellular receptors for DENV, Vav1 was identified as a virus-binding partner in mosquito cells and proposed to be involved intracellularly in facilitating DENV entry (33). In the study here, the expression of Vav mRNA and proteins following DENV infection in monocyte-derived macrophages (MDMs), a key target for DENV replication in vivo, and ARPE-19 cells, which are relevant to DENV-mediated ocular inflammation, have been defined. ...
Vav proteins are guanine exchange factors that activate Rac1/RhoA signaling to regulate many biological processes including inflammatory responses. Here, the expression and functional significance of Vav’s were investigated during dengue virus (DENV) infection. In primary monocyte-derived macrophages, Vav1, −2, and −3 mRNA levels demonstrate variable responses to DENV infection and correlate with DENV-induced host inflammatory (IL-6 and TNF-α), antiviral (viperin), or cell adhesion [intercellular cell adhesion molecule (ICAM-1)] mRNA induction. Strong positive correlations were seen in particular for Vav2 with TNF-α and Vav3 with IL-6 mRNA. In the retinal pigmented epithelial cell line, ARPE-19, Vav2 was the main Vav expressed and was not affected by DENV infection. Heterologous Vav1 expression in ARPE-19 cells induced an increase in basal IL-6 mRNA but did not enhance DENV-induced mRNA responses. DENV RNA and DENV-induced viperin and IL-6 mRNA responses were also unaffected by Vav2 siRNA knockdown. Treatment of DENV-infected ARPE-19 cells with EHop-016 to block Vav signaling did not affect DENV RNA levels but increased DENV-mediated induction of IL-6 mRNA. More detailed assessment of DENV-induced responses to azathioprine, a clinically used immunosuppressant that can also block Vav signaling and act as a nucleoside analog, similarly demonstrated no change in DENV RNA levels but resulted in inhibition of DENV-induced phospho-ERK and increased DENV-induced-IL-6 and viperin mRNA in ARPE-19 cells. Thus, levels of Vav are associated with DENV-induced inflammatory responses, and blocking Vav signaling pathways does not compromise control of viral replication but may influence DENV-induced host responses.
IMPORTANCE
Dengue disease is characterized by an inflammatory-mediated immunopathology, with elevated levels of circulating factors including TNF-α and IL-6. If the damaging inflammatory pathways could be blocked without loss of antiviral responses or exacerbating viral replication, then this would be of potential therapeutic benefit. The study here has investigated the Vav guanine exchange factors as a potential alternative signaling pathway that may drive dengue virus (DENV)-induced inflammatory responses, with a focus on Vav1 and 2. While Vav proteins were positively associated with mRNA for inflammatory cytokines, blocking Vav signaling didn’t affect DENV replication but prevented DENV-induction of p-ERK and enhanced IL-6 (inflammatory) and viperin (antiviral) mRNA. These initial data suggest that Vav proteins could be a target that does not compromise control of viral replication and should be investigated further for broader impact on host inflammatory responses, in settings such as antibody-dependent enhancement of infection and in different cell types.
... Previous reports have demonstrated that the catalytic β-subunit of the F1 domain of ATP synthase mediates the infectious entry of some viruses by acting as a cell surface receptor protein (Fongsaran et al. 2014;Paingankar et al. 2010). It should be remarked that although in the past the F 1 F 0 -ATP synthase was thought to be limited to the inner mitochondrial membrane, new evidence has found that this enzyme complex can also be found on the cell surface of a variety of normal (endothelial, hepatocytes, adipocytes) and tumor cells (Das et al. 1994;Kim et al. 2004;Martinez et al. 2003;Moser et al. 1999). ...
The white spot syndrome virus (WSSV) is a highly lethal pathogen for several crustacean species, which implies that this virus has caused high mortalities and significant economic losses for the global shrimp aquaculture industry. Viruses’ entry into the host cell depends on the interaction between those proteins located on the surface of the virus and its attachment to specific surface-exposed cell receptors. In this review, the findings in the interaction of the receptors recognized by WSSV proteins are summarized and discussed, providing thus a comprehensive and finer understanding of the importance of these molecules for WSSV pathogenesis, as well as suggesting strategies targeting critical components for the design and development of therapeutic antivirals against WSSV that could be used in shrimp farming.
... Reductions in PCLV led to increases in DENV replication at the genomic RNA level and virion production suggesting that PCLV could contribute to inhibition of DENV in Aag2.tet cells. We then explored the expression levels of a number of genes whose products have been shown to be involved in virus-host interactions and immunity: Dicer-2, the enzyme involved in the RNAi response (antiviral) (Blair and Olson, 2014); exoribonuclease 1 (XRN1), involved in degradation of flaviviral genomic RNA (Funk et al., 2010), but also shown to contribute in Wolbachia virus blocking phenotype (Thomas et al., 2018); cecropin, involved in insect immunity and potentially anti-viral response (Tikhe and Dimopoulos, 2021); prohibitin, which has been shown to bind to DENV (Paingankar et al., 2010); and kinesin, a cytoskeleton motor protein shown to be actively involved in the life cycles of various viruses (Tati and Alisaraie, 2022). Results showed that the two antiviral genes, Dicer-2 and XRN1, were significantly upregulated and the anti-microbial peptide cecropin significantly downregulated in both Aag2.wAlbB and Aag2.tet cells compared to Aag2 cells. ...
Wolbachia pipientis is known to block replication of positive sense RNA viruses. Previously, we created an Aedes aegypti Aag2 cell line (Aag2.wAlbB) transinfected with the wAlbB strain of Wolbachia and a matching tetracycline-cured Aag2.tet cell line. While dengue virus (DENV) was blocked in Aag2.wAlbB cells, we found significant inhibition of DENV in Aag2.tet cells. RNA-Seq analysis of the cells confirmed removal of Wolbachia and lack of expression of Wolbachia genes that could have been due to lateral gene transfer in Aag2.tet cells. However, we noticed a substantial increase in the abundance of phasi charoen-like virus (PCLV) in Aag2.tet cells. When RNAi was used to reduce the PCLV levels, DENV replication was significantly increased. Further, we found significant changes in the expression of antiviral and proviral genes in Aag2.tet cells. Overall, the results reveal an antagonistic interaction between DENV and PCLV and how PCLV-induced changes could contribute to DENV inhibition.
... Initially found at the mitochondrion surface, the ATP synthase was also identified as a cell membrane protein in many cell types, including in Ae. aegypti cells [59]. In studies to identify candidate receptors for DENV-2 in the Aedes midgut, ATP synthase β(ATPSβ) was suggested to function as an ATP provider facilitating the function of the HSc 70 chaperon, which could help to accumulate virus particles on the membrane [60]. The analysis of the role of ATPSβ in the CHIKV infection of Ae. ...
Mosquito-borne diseases caused by viruses and parasites are responsible for more than 700 million infections each year. Anopheles and Aedes are the two major vectors for, respectively, malaria and arboviruses. Anopheles mosquitoes are the primary vector of just one known arbovirus, the alphavirus o’nyong-nyong virus (ONNV), which is closely related to the chikungunya virus (CHIKV), vectored by Aedes mosquitoes. However, Anopheles harbor a complex natural virome of RNA viruses, and a number of pathogenic arboviruses have been isolated from Anopheles mosquitoes in nature. CHIKV and ONNV are in the same antigenic group, the Semliki Forest virus complex, are difficult to distinguish via immunodiagnostic assay, and symptomatically cause essentially the same human disease. The major difference between the arboviruses appears to be their differential use of mosquito vectors. The mechanisms governing this vector specificity are poorly understood. Here, we summarize intrinsic and extrinsic factors that could be associated with vector specificity by these viruses. We highlight the complexity and multifactorial aspect of vectorial specificity of the two alphaviruses, and evaluate the level of risk of vector shift by ONNV or CHIKV.
... But for a wide variety of normal cells, including endothelial cells, adipocytes, keratinocytes, and hepatic cells, active ATP synthase activity on the cell surface has been documented 41 . Fongsaran et al.(2014) reported that ATPSβ plays a role in Chikungunya virus (CHIKV) infection of mosquito cells 41 , and there is evidence showing that the same protein is involved in the internalization of white spot syndrome virus (WSSV) to shrimp cells 42 , and possibly binding of dengue virus to insect cells 43 , suggests that this is a conserved virus-arthropod interaction. Therefore, by targeting ATP8_AE-DAE, the interaction of the pathogens and the mosquito vector can be disrupted and thereby, transmission to the human host may be effectively prevented. ...
Aedes is an important vector for various viruses that cause dengue, chikungunya and zika, which affect human health globally. Due to regular outbreaks of these diseases worldwide, there is a need to identify essential vector proteins that are critical for the survival of the vector, which may be targeted to control the spread of vector-borne disease (VBD). In silico computational methods involving comparative proteomics, analysis of orthologous proteins common amongst members of Aedes genus and protein-protein interaction (PPI) pathway were used to identify essential proteins that could act as novel therapeutic candidates. Twenty-three conserved proteins between A. aegypti and A. albopictus were identified from a BLASTP search with an e-value threshold of 0.005, and their PPI networks were constructed in the STRING database. The merged network was analyzed using various Cytoscape plugins viz. ClusterONE, Cytohubba and MCODE. Thirty-one hub proteins were identified from the system's network biology analysis, and detailed data and literature mining were carried out. Twelve novel vector-control target proteins of A. aegypti, having no human homologs, were determined in the present study that can effectively act as potential therapeutic candidates for drug design and vaccine development.
... In addition, 10 mpi with DENV, the co-localization of small groups of viral particles with host actin was detected, suggesting that actin filaments may favor movement of the viral particles [75]. At 48 hpi, transcription of actin and tubulin genes decreases to increase again at 96 hpi, suggesting a viral-mediated host shutoff effect followed by the activation of mechanisms that maintain homeostasis and reorganization of cytoskeleton during virus infection [78]. Further evidence on DENV manipulation of the host cell cytoskeleton comes from proteomic and transcriptomic analyses of different developmental stages of the mosquito Ae. aegypti. ...
Viruses are excellent manipulators of host cellular machinery, behavior, and life cycle, with the host cell cytoskeleton being a primordial viral target. Viruses infecting insects generally enter host cells through clathrin-mediated endocytosis or membrane fusion mechanisms followed by transport of the viral particles to the corresponding replication sites. After viral replication, the viral progeny egresses toward adjacent cells and reaches the different target tissues. Throughout all these steps, actin and tubulin re-arrangements are driven by viruses. The mechanisms used by viruses to manipulate the insect host cytoskeleton are well documented in the case of alphabaculoviruses infecting Lepidoptera hosts and plant viruses infecting Hemiptera vectors, but they are not well studied in case of other insect–virus systems such as arboviruses–mosquito vectors. Here, we summarize the available knowledge on how viruses manipulate the insect host cell cytoskeleton, and we emphasize the primordial role of cytoskeleton components in insect virus motility and the need to expand the study of this interaction.
... However, the exit of HSC70 from nucleus to cytoplasm is inhibited upon stress, limiting its function to the nuclear compartment (Kodiha et al., 2005). Meanwhile, studies have reported that HSC70 is also a membrane-anchored protein (Arispe et al., 2002), and it has been proved to be expressed on the cytoplasmic membrane of several different cell lines, such as MA104, Caco-2, Hep2, BHK , C6/36 (Paingankar et al., 2010), B cells (Mayer, 2005), and the suckling mouse intestinal epithelial cells (Guerrero and Moreno, 2012), and participates in virus invasion as receptor or co-receptor. ...
Heat shock cognate 71-kDa protein (HSC70), a constitutively expressed molecular chaperon within the heat shock protein 70 family, plays crucial roles in maintaining cellular environmental homeostasis through implicating in a wide variety of physiological processes, such as ATP metabolism, protein folding and transporting, antigen processing and presentation, endocytosis, and autophagy. Notably, HSC70 also participates in multiple non-communicable diseases and some pathogen-caused infectious diseases. It is known that virus is an obligatory intracellular parasite and heavily relies on host machineries to self-replication. Undoubtedly, HSC70 is a striking target manipulated by virus to ensure the successful propagation. In this review, we summarize the recent advances of the regulatory mechanisms of HSC70 during viral infections, which will be conducive to further study viral pathogenesis.
... These preliminary results seem to demonstrate an in vivo role in phytoplasma infection mechanism for ATP synthase β. Interestingly this protein is already known to interact in vitro with pathogen membrane proteins (Galetto et al., 2011;Paingankar et al., 2010). ...
... It was also found that the ATP synthase subunit is present on the cell surface where it may serve as a cell membrane receptor [51]. On the other hand, ATP synthase has been identified as a virusinteraction protein capable of mediating the entry of virus into host cells and acts as a factor that mediates human immunodeficiency virus-1 (HIV-1) transfer between antigenpresenting cells and CD4 + target cells [52][53][54][55]. The rapid replication of DTMUV in host cells will consume abundant host ATP and impact other energy-dependent biological functions of the host cells. ...
Outbreaks of duck Tembusu virus (DTMUV) have caused substantial economic losses in the major duck-producing regions of China since 2010. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in duck embryo fibroblast cells (DEFs) infected and mock-infected with DTMUV. In total, 434 cellular proteins were differentially expressed, among which 116, 76, and 339 proteins were differentially expressed in the DTMUV-infected DEFs at 12, 24, and 42 hours postinfection, respectively. The Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and cellular organismal processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding and catalytic activity. Some selected proteins that were found to be differentially expressed in DTMUV-infected DEFs were further confirmed by real-time PCR. The results of this study provide valuable insight into DTMUV-host interactions. This could lead to a better understanding of DTMUV infection mechanisms.
... Microtubules and the cytoskeleton have been intensively studied and found to be involved in several steps of the flaviviral replication cycle, such as viral entry,i ntracellular transport, virion assembly,a nd egress. [8,9] Although the involvement of microtubules in cell entry of DENV has been reported to be crucial, [10,11] other reports suggest am icrotubule-independent cell entry of differentD ENV serotypes. [12] Also in thesec ases, microtubules might be an onessential, but nevertheless supportivefactor for the infection of cells. ...
Recent studies indicate that tubulin can be a host factor for vector‐borne flaviviruses like dengue (DENV) and Zika (ZIKV), and inhibitors of tubulin polymerization such as colchicine have been demonstrated to decrease virus replication. However, toxicity limits the application of these compounds. Herein we report prodrugs based on combretastatin and colchicine derivatives that contain an ester cleavage site for human carboxylesterase, a highly abundant enzyme in monocytes and hepatocytes targeted by DENV. Relative to their parent compounds, the cytotoxicity of these prodrugs was reduced by several orders of magnitude. All synthesized prodrugs containing a leucine ester were hydrolyzed by the esterase in vitro. In contrast to previous reports, the phenylglycine esters were not cleaved by human carboxylesterase. The antiviral activity of combretastatin, colchicine, and selected prodrugs against DENV and ZIKV in cell culture was observed at low micromolar and sub‐micromolar concentrations. In addition, docking studies were performed to understand the binding mode of the studied compounds to tubulin.
... The functional rotavirus receptor is a complex of several cell surface molecules that include HSC 70 [22]. Dengue, a flavivirus, was reported to interact with the HSC 70 protein among other housekeeping proteins for its entry into mosquito cells [32]. Further, HSC 70 has been proposed as a penetration receptor mediating Japanese encephalitis virus entry into C6/36 cells [23]. ...
Aim:
The study was designed to identify putative Chikungunya virus (CHIKV) receptor/s on C6/36 cells that facilitate viral entry.
Methods:
The virus overlay protein binding assay (VOPBA) was adopted to identify CHIKV-interacting bands present in C6/36 cell membrane and identity of the protein was established by mass spectrometry. The role of this protein as a putative CHIKV receptor on C6/36 cells was confirmed by infection inhibition assay. Cell surface localization of the identified protein was studied by indirect immunofluorescence assay (IFA) on nonpermeabilized cells and by flow cytometry. Interaction between this protein and CHIKV was confirmed by co-immunoprecipation (Co-IP) and Western blotting. The effect of depletion of the identified protein by quercetin was demonstrated by infection inhibition assay.
Results:
A 70-kDa protein was identified as a CHIKV-interacting protein by VOPBA. MALDI-TOF analysis followed by homology search revealed that this protein could be heat shock cognate 70 (HSC 70). Anti-HSC 70 antibodies blocked CHIKV entry into C6/36 cells in a dose-dependent manner. IFA and flow cytometry analysis demonstrated HSC 70 localization on C6/36 cell surface. Co-IP experiments confirmed the interaction between HSC 70 and CHIKV envelope. Quercetin- and YM-01-treated C6/36 cells exhibited dose-dependent infection inhibition.
Conclusion:
HSC 70 serves as a putative CHIKV receptor on C6/36 cells.
... Some PHB1 proteins have been reported to interact with low density detergent-insoluble lipid raft domains in the plasma membrane 26,51 , acting as transmembrane adapters to activate downstream signals 14,52,53 . Previous studies also identified that PHB1 on plasma membrane acts as a viral receptor protein to facilitate virus entry into host cells 8,54,55 . In addition, studies found that expression level of PHB1 on T cell surfaces is significantly upregulated when T cells are activated 56 . ...
Prohibitin (PHB) was originally isolated and characterized as an anti-proliferative gene in rat liver. The evolutionarily conserved PHB gene encodes two human protein isoforms with molecular weights of ~33 kDa, PHB1 and PHB2. PHB1 and PHB2 belong to the prohibitin domain family, and both are widely distributed in different cellular compartments such as the mitochondria, nucleus, and cell membrane. Most studies have confirmed differential expression of PHB1 and PHB2 in cancers compared to corresponding normal tissues. Furthermore, studies verified that PHB1 and PHB2 are involved in the biological processes of tumorigenesis, including cancer cell proliferation, apoptosis, and metastasis. Two small molecule inhibitors, Rocaglamide (RocA) and fluorizoline, derived from medicinal plants, were demonstrated to interact directly with PHB1 and thus inhibit the interaction of PHB with Raf-1, impeding Raf-1/ERK signaling cascades and significantly suppressing cancer cell metastasis. In addition, a short peptide ERAP and a natural product xanthohumol were shown to target PHB2 directly and prohibit cancer progression in estrogen-dependent cancers. As more efficient biomarkers and targets are urgently needed for cancer diagnosis and treatment, here we summarize the functional role of prohibitin domain family proteins, focusing on PHB1 and PHB2 in tumorigenesis and cancer development, with the expectation that targeting the prohibitin domain family will offer more clues for cancer therapy.
... Reproduced in part from Fruttero et al. (2011), with permission is its role as the receptor of apolipoprotein A-I (apoA-I) in the reverse cholesterol transport mediated by high-density lipoproteins (HDL) (Vantourout et al. 2010). Ecto-ATPases were also reported in arthropods, fulfilling roles as binding proteins that mediate diverse biological functions, including hormonal regulation (Zalewska et al. 2009), phytoplasma transmission specificity (Galetto et al. 2011), hematopoiesis (Lin et al. 2009), and virus acquisition (Paingankar et al. 2010;Fongsaran et al. 2014). More recently, it was reported that ecto-β-ATPase in P. megistus was involved in bidirectional lipophorin-mediated lipid transfer to the enterocytes . ...
In insect physiology, the mechanisms involved in the buildup and regulation of yolk proteins in developing oocytes have been thoroughly researched during the last three decades. Comparatively, the study of lipid metabolism in oocytes had received less attention. The importance of this issue lies in the fact that lipids make up to 40% of the dry weight of an insect egg, being the most important supply of energy for the developing embryo. Since the oocyte has a very limited capacity to synthesize lipids de novo, most of the lipids in the mature eggs arise from the circulation. The main lipid carriers in the insect circulatory system are the lipoproteins lipophorin and vitellogenin. In some species, the endocytosis of lipophorin and vitellogenin may account for about 10% of the lipids present in mature eggs. Thus, most of the lipids are transferred by a lipophorin-mediated pathway, in which the lipoprotein unloads its lipid cargo at the surface of oocytes without internalization. This chapter recapitulates the current status on lipid storage and its utilization in insect oocytes and discusses the participation of key factors including lipoproteins, transfer proteins, lipolytic enzymes, and dynamic organelles such as lipid droplets. The new findings in the field of lipophorin receptors are presented in the context of lipid accumulation during egg maturation, and the roles of lipids beyond energy source are summarized from the perspective of oogenesis and embryogenesis. Finally, prospective and fruitful areas of future research are suggested.
... Aed a 8 is a member of the Heat-shock protein-70 (Hsp70) family, which are exploited by dengue virus35 and mosquito-borne flaviviruses (encephalitis virus)36 to attach and penetrate mosquito cells. Hsp70 allergens have been reported in cockroach37 and D. farinae.18 ...
Background:
Saliva and muscle derived mosquito allergens have been purified and characterized. However, the complete set of allergens remains to be elucidated. In this study, we identified and characterized IgE-binding proteins from the mosquito species A. aegypti.
Methods:
Serum was obtained from 15 allergic individuals with asthma and/or rhinitis and sensitized to mosquito. IgE-binding was determined by ELISA. Total proteins from freeze dried bodies of A. aegypti were extracted and IgE reactive proteins were identified by 2D-gel electrophoresis, followed by Western blot with pooled or individual sera. IgE-reactive spots were further characterized by mass spectrometry.
Results:
Twenty five IgE-reactive spots were identified, corresponding to 10 different proteins, some of which appeared as different variants or isoforms. Heat shock cognate 70 (HSC-70) and Tropomyosin showed IgE reactivity with 60% of the sera, Lysosomal aspartic protease and "AAEL006070-PA" (Uniprot: Q177P3) with 40% and the other proteins with less than 33.3% of the sera. Different variants, or isoforms of Tropomyosin, Arginine or Creatine kinase, Glyceraldehyde-3-phosphate dehydrogenase (GPDH), Calcium-binding protein and Phosphoglycerate mutase were also identified. The mixture of three allergens (Aed a 6, Aed a 8 and Aed a 10) seems to identify more than 80% of A. aegypti sensitized individuals, indicating that these allergens should be considered when designing of improved mosquito allergy diagnostic tools.
Conclusions:
The newly identified allergens may play a role in the pathophysiology of mosquito allergy in the Tropics, and some of them might be important arthropod-related proteins involved in cross-reactivity between A. aegypti and other allergenic arthropods. This article is protected by copyright. All rights reserved.
... In several hemipterans, vectors of phytoplasmas, ectopic aand b-ATPase of midgut and salivary gland cells were capable of binding antigenic proteins, thus controlling the transmission specificity of these phytopathogenic bacteria (Galetto et al., 2011). Plasma membrane b-ATPase was also involved in the acquisition of dengue virus (Paingankar et al., 2010) and chikungunya virus (Fongsaran et al., 2014) by the mosquito Aedes sp. These reports strengthened the attribution of plasma membrane b-ATPase as a binding protein and showed its multiple and relevant functions. ...
Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic β chain of ATP synthase (β-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of β-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of β-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of β-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and β-ATPase. In vivo experiments with an anti-β-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that β-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus, similarly to its function in the midgut of such a vector. In addition, the role of β-ATPase as a docking receptor seems to be independent of the enzymatic ATP synthase activity.
... An effective TBV for DENV should be able to block infection of all serotypes to prevent an increase in the severity of cases. Recent research has discovered several mosquito molecules with a critical role in DENV cell entry and replication [98,99]. As seen in WNV, several C-type lectins have been associated with Ae. aegypti and DENV infection. ...
Infectious diseases caused by arboviruses (viruses transmitted by arthropods) are undergoing unprecedented epidemic activity and geographic expansion. With the recent introduction of West Nile virus (1999), chikungunya virus (2013) and Zika virus (2015) to the Americas, stopping or even preventing the expansion of viruses into susceptible populations is an increasing concern. With a few exceptions, available vaccines protecting against arboviral infections are nonexistent and current disease prevention relies on vector control interventions. However, due to the emergence of and rapidly spreading insecticide resistance, different disease control methods are needed. A feasible method of reducing emerging tropical diseases is the implementation of vaccines that prevent or decrease viral infection in the vector. These vaccines are designated ?transmission blocking vaccines?, or TBVs. Here, we summarize previous TBV work, discuss current research on arboviral TBVs and present several promising TBV candidates.
... ATP synthase and ATPase are reported to have a role in the viral infection process of different pathosystems, such as white spot syndrome or whispovirus in Pacific white shrimp (Litopenaeus vannamei), and Nucleopolyhedrovirus in silkworm (Bombyx mori) (Liang et al., 2010;Lu et al., 2013). In the well-studied mosquito vector, A. aegypti, ATP synthase is found to be part of a complex network associated with the brush border surface of the midgut, which also includes actin and carbohydrates, and it may be implicated in the pathogen transmission (Zieler et al., 2000;Paingankar et al., 2010). Several proteomic studies report that bacterial pathogens interact with the surface proteins of actin, Fig. 6. ...
The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two-dimensional gel electrophoresis (2-DE). A total of 1170 spots are detected in the 2-DE gels of the three subcellular fractions. One hundred and sixty-four differentially expressed proteins are successfully identified using liquid chromatography-dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.
... Table 2 lists the mosquito proteins that putatively bound CRVP379 and were not identified during control experiments. One of the proteins identified, prohibitin, was previously characterized as binding to DENV in Aedes A7 cells [56] and has also been suggested as a putative DENV receptor in mosquito cells [57]. ...
Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.
... There is evidence that DENV interacts initially with heparan sulfate proteoglycans essential in anchoring at the cell surface [49,50]. Several target proteins on the cell surface have been identified and clathrinmediated endocytosis has been shown to play role in DENV internalization in both mammalian and insect cells [48,[51][52][53]. It is possible that viruses have evolved to use abundant LDL and LRP receptors to gain ubiquitous access to human tissues. ...
It is established that cell entry of low density lipoprotein particles (LLPs) containing Apo B100 and Apo E is mediated by receptors and GAGs. Receptor ligand motifs, XBBBXXBX, XBBXBX, and ΨBΨXB, and mono- and bipartite NLS sequences are abundant in Apo E and Apo B100 as well as in envelope and capsid proteins of dengue viruses 1-4 (DENV1-4). Synthetic, fluorescence-labeled peptides of sequences in DENV2 envelope protein, and DENV3 capsid that include these motifs were used to conduct a qualitative assessment of cell binding and entry capacity using HeLa cells. DENV2 envelope peptide, Dsp2EP, 0564Gly-Gly0595, was shown to bind and remain at the cell surface. In contrast, DENV3 capsid protein peptide, Dsp3CP, 0002Asn-Gln0028, readily enters HeLa cells and accumulates at discrete loci in the nucleus. FITC-labeled dengue synthetic peptides colocalize with low density lipoprotein-CM-DiI and Apo E-CM-DiI to a degree suggesting that dengue viruses may utilize cell entry pathways used by LLPs.
... Interestingly, involvement of ATPsyn β in the entry of chikungunya virus (CHIKV) into insect cells has been recently demonstrated, and showed a significant reduction in viral entry and virus production by both antibody inhibition and siRNA-mediated down regulation experiments targeted to ATPsyn β [13]. Our current results revealed the evidence that the same protein, ATPsyn β, involved in the infection of WSSV to shrimp, and possibly CHIKV and dengue virus to insect cells as well [13,14]. These informations will lead us a better illustration of the entry process of WSSV into the host cells, as well as the dengue virus into insect cells. ...
Background
Over the past a few years, evidences indicate that adenosine triphosphate (ATP) is an energy source for the binding, maturation, assembly, and budding process of many enveloped viruses. Our previous studies suggest that the F1-ATP synthase beta subunit (ATPsyn β, BP53) of the shrimp Litopenaeus vannamei (L. vannamei) might serve as a potential receptor for white spot syndrome virus (WSSV)’s infection.
Methods
BP53 was localized on the surface of shrimp hemocytes and gill epithelial cells by immunofluorescence assay and immunogold labeling technique. Cell surface ATP synthesis was demonstrated by an in vitro bioluminescent luciferase assay. Furthermore, the expression of bp53 after WSSV infection was investigated by RT-PCR test. In addition, RNAi was developed to knock down endogenous bp53.
Results
BP53 is present on shrimp cell surface of hemocytes and gill epithelia. The synthesized ATP was detectable in the extracellular supernatant by using a bioluminescence assay, and the production declined post WSSV binding and infection. Knocking down endogenous bp53 resulted in a 50% mortality of L. vannamei.
Conclusion
These results suggested that BP53, presenting on cell surface, likely served as one of the receptors for WSSV infection in shrimp. Correspondingly, WSSV appears to disturb the host energy metabolism through interacting with host ATPsyn β during infection. This work firstly showed that host ATP production is required and consumed by the WSSV for binding and proceeds with infection process.
... These findings could indicate that under conditions of coinfections in cell lines, predominant replication of one serotype could be due to the presence of specific receptors that allow entry, as previously explained. For example, a lamininbinding protein has been described as being responsible for the entry of serotype 3 [33], while the entry of serotype 2 into mosquito cells has been linked to Hsc 70 proteins (shock cognate protein 70) [39] and prohibitin [32]. So far, mechanisms that relate to differences in replication once the virus has entered the cell have not been described. ...
Introduction:
Different dengue virus (DENV) serotypes have been associated with greater epidemic potential. In turn, the increased frequency in cases of severe forms of dengue has been associated with the cocirculation of several serotypes. Because Colombia is a country with an endemic presence of all four DENV serotypes, the aim of this study was to evaluate the in vivo and in vitro replication of the DENV-2 and DENV-3 strains under individual infection and coinfection conditions.
Methodology:
C6/36HT cells were infected with the two strains individually or simultaneously (coinfection). Replication capacity was evaluated by RT-qPCR, and the effects on cell viability were assessed with an MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Additionally, Aedes aegypti mosquitoes were artificially fed the two strains of each serotype individually or simultaneously. The viral genomes were quantified by RT-qPCR and the survival of the infected mosquitoes was compared to that of uninfected controls.
Results:
In single infections, three strains significantly affected C6/36HT cell viability, but no significant differences were found in the replication capacities of the strains of the same serotype. In the in vivo infections, mosquito survival was not affected, and no significant differences in replication between strains of the same serotype were found. Finally, in coinfections, serotype 2 replicated with a thousandfold greater efficiency than serotype 3 did both in vitro and in vivo.
Conclusions:
Due to the cocirculation of serotypes in endemic regions, further studies of coinfections in a natural environment would further an understanding of the transmission dynamics that affect DENV infection epidemiology.
... They are located ubiquitously in cytoplasm, mitochondria, nucleus and plasma membrane [86][87][88]. Independently, another group also found HSC70 interacting with DENV2 in C6/36 and A7 cells as well as in Ae aegypti females [75]. Furthermore, the chaperone HSC70 has been implicated in JEV entry into C6/36 [89] and mammalian cells [90]. ...
Viral entry into host cells primordially defines tropism and represents an attractive target to counteract infection either by antiviral agents or by immune mediated mechanisms. Research on Dengue virus entry presents interesting challenges. Whatever the mechanism dengue virus exploits to gain entry into cells, this had to be evolutionarily conserved, so that it is now present in arthropod and human cells. Until now, dengue cellular receptors were not completely unraveled. However, we have clues about the key steps dengue virus is relying on. Initially a group of factors that interact with the virus through carbohydrate interaction assure its adherence and further contact with a protein receptor complex, which is held together thanks to its special interaction with cell membrane lipidic platforms. This interaction may be so intimate that it may trigger not only viral entry through receptor-mediated endocytosis, but also activation of cell signaling pathways that the virus is going to subvert to its advantage.
Flaviviruses cause numerous pathologies in humans across a broad clinical spectrum with potentially severe clinical manifestations, including hemorrhagic and neurological disorders. Among human flaviviruses, some viral proteins show high conservation and are good candidates as targets for drug design. From an epidemiological point of view, flaviviruses cause more than 400 million cases of infection worldwide each year. In particular, the Yellow Fever, dengue, West Nile, and Zika viruses have high morbidity and mortality—about an estimated 20,000 deaths per year. As they depend on human vectors, they have expanded their geographical range in recent years due to altered climatic and social conditions. Despite these epidemiological and clinical premises, there are limited antiviral treatments for these infections. In this review, we describe the major compounds that are currently under evaluation for the treatment of flavivirus infections and the challenges faced during clinical trials, outlining their mechanisms of action in order to present an overview of ongoing studies. According to our review, the absence of approved antivirals for flaviviruses led to in vitro and in vivo experiments aimed at identifying compounds that can interfere with one or more viral cycle steps. Still, the currently unavailability of approved antivirals poses a significant public health issue.
ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase alpha and beta family, including the alpha and beta subunits of ATP synthase (RpATPSynA and RpATPSynB), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organs, being their expression highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynB knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynB increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynA knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.
Dengue virus type Ⅱ (DENV2) is a primary serotype responsible for the dengue fever epidemic, and Aedes aegypti is the main DENV2 vector. Understanding the Aedes aegypti immune mechanism against DENV2 is the basis for research on immune blockade in mosquitoes. Some preliminary studies lack validation in the literature, so this study was performed to further study and validate the potential target genes to provide a further basis for screening key target genes. We screened 51 genes possibly related to Aedes aegypti infection and immunity from the literature for further verification. First, bioinformatic methods such as GO, KEGG and PPI analysis were used, and then RT–qPCR was used to detect the changes in mRNA expression in the midguts and salivary glands of Aedes aegypti infected with DENV2.Bioinformatic analysis showed that mostly genes of the glucose metabolism pathway and myoprotein were influenced. In salivary glands, the Gst (xa) and Toll (xb) expression levels were significantly correlated with DENV2 load (y, lg[DENV2 RNA copies]), y= -3436xa+0.2287xb+3.8194 (adjusted R² = 0.5563, F = 9.148, PF = 0.0045). In midguts, DENV2 load was significantly correlated with the relative Fba(R² = 0.4381, t=2.497, p<0.05, df=8), UcCr(R² = 0.4072, t=2.344, p<0.05, df=8) and Gbps1(R² = 0.4678, t=2.652, p<0.05, df=8) expression levels, but multiple regression did not yield significant results. This study shows that genes related to glucose metabolism and muscle proteins contribute to the interaction between Aedes aegypti and dengue virus. It was confirmed that SAAG-4, histone H4, endoplasmin, catalase and other genes are involved in the regulation of DENV2 infection in Aedes aegypti. It was revealed that GST and Toll in salivary glands may have antagonistic effects on the regulation of DENV2 load. Fba, UcCr and Gbps1 in the midgut may increase DENV2 load. These study results further condensed the potential target gene range of the Aedes aegypti immune mechanism against DENV2 infection and provided basic information for research on the Aedes aegypti in vivo blockade strategy against DENV2.
Aedes aegypti is inherently susceptible to arboviruses. The geographical expansion of this vector host species has led to the persistence of Dengue, Zika and Chikungunya human infections. These viruses take advantage of the mosquito’s cell to create an environment conducive for their growth. Arboviral infection triggers transcriptomic and protein dysregulation in Ae. aegypti and in effect, host antiviral mechanisms are compromised. Currently, there are no existing vaccines able to protect human hosts from these infections and thus, vector control strategies such as Wolbachia mass release program is regarded as a viable option. Considerable evidence demonstrates how the presence of Wolbachia interferes with arboviruses by decreasing cellular components vital for the pathogen and strengthening antiviral host responses. However, variation in the magnitude of Wolbachia’s viral inhibition that is neither due to strain nor density has been observed. Furthermore, the cellular mechanisms involved in the endosymbiont’s pathogen-blocking differs among hosts. This prompts the need to explore the cellular interactions between Ae. aegypti-arboviruses-Wolbachia and how these interactions overall affect the mosquito’s cell. Understanding what happens at the cellular and molecular level will provide evidence on the sustainability of Wolbachia vector control.
Chikungunya virus (CHIKV) is an arthropod-borne virus that has re-emerged recently and has spread to previously unaffected regions, resulting in millions of infections worldwide. The genus Alphavirus, in the family Togaviridae, contains several members with a similar potential for epidemic emergence. In order for CHIKV to replicate in targeted cell types it is essential for the virus to enter these cells. In this review, we summarize our current understanding of the versatile and promiscuous steps in CHIKV binding and entry into human and mosquito host cells. We describe the different entry pathways, receptors, and attachment factors so far described for CHIKV and other mosquito-borne alphaviruses and discuss them in the context of tissue tropism and potential therapeutic targeting.
As traditional approaches to the control of dengue and Zika are insufficient, significant efforts have been made to develop utilization of the endosymbiotic bacterium Wolbachia to reduce the ability of mosquitoes to transmit pathogens. Although Wolbachia is known to inhibit flaviviruses in mosquitoes, including dengue virus (DENV) and Zika virus (ZIKV), it remains unclear how the endosymbiont interferes with viral replication cycle. In this study, we have carried out viral binding assays to investigate the impact of the Wolbachia strain wAlbB on the attachment of DENV serotype 2 (DENV-2) and ZIKV to Aedes aegypti Aag-2 cells. RNA interference (RNAi) was used to silence a variety of putative mosquito receptors of DENV that were differentially regulated by wAlbB in Aag-2 cells, in order to identify host factors involved in the inhibition of viral binding. Our results showed that, in addition to suppression of viral replication, Wolbachia strongly inhibited binding of both DENV-2 and ZIKV to Aag-2 cells. Moreover, the expression of two putative mosquito DENV receptors – dystroglycan and tubulin – was downregulated by wAlbB, and their knock-down resulted in the inhibition of DENV-2 binding to Aag-2 cells. These results will aid in understanding the Wolbachia-DENV interactions in mosquito and the development of novel control strategies for mosquito-borne diseases.
Dengue, the most prevalent arboviral disease worldwide, is caused by any of the four dengue virus (DENV) serotypes that co-circulate constantly in hyperendemic areas such as Medellin (Colombia), and these serotypes are transmitted by mosquitoes of the genus Aedes. In this study, we evaluated the replicative capacity of strains isolated in Medellin between 2003 and 2007 in C6/36 cells and in colonies of Aedes aegypti collected during 2010-2011 from high or low-incidence areas within the same city. The phylogenetic analysis grouped isolates according to the predominant genotypes found in the Americas, and the in vitro characterization showed differences in the morphological changes induced by the isolates of each of the isolated serotypes compared to the reference serotypes. In vitro replicative capacity studies demonstrated that genomic copy number increased at four days post-infection and that cell viability decreased significantly compared to the control for all serotypes. The largest number of genomic copies in C6/36 was produced by DENV-2, followed by DENV-1 and DENV-4; DENV-3 produced the smallest number of genomic copies and had the smallest negative effect on cell viability. Finally, differences in the in vivo replication of intercolonial serotypes between the Rockefeller colony and the field colonies and among the intracolonial serotypes were found. The replication of DENV-2 at 7 and 14 days in both high- and low-incidence colonies was higher than that of the other serotypes, and replication of DENV-3 in the mosquito colonies was the most stable on the days evaluated. Our results support the notion that replication and, possibly, DENV transmission and severity depend on many factors, including serotype and vector characteristics.
Dengue virus (DENV) infection is a mosquito-borne disease and remains one of the major public health problems worldwide, particularly in tropical and sub-tropical regions. In most cases, the infection causes only mild and self-limiting illness, namely dengue fever (DF). The infection, however, can develop to severe and life-threatening disease known as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Recent studies have suggested that host factors, while protect against the invaded virus, can be modulated and utilized by DENV for its survival and propagation within host cells, leading to infection. Heat shock protein 70 (HSP70) family is one of the key components of host machinery for protein homeostasis (proteostasis). Molecular chaperone function of HSP70 family is achieved by cooperative networking of HSP70 family members (HSP70s) and their co-chaperones to regulate ATP-ADP cycling. According to their fundamental importance, HSP70s have been discovered as the host factors hijacked by many viruses, including DENV, for their efficient infection. This chapter provides the current knowledge on how DENV manipulates host HSP70s in multiple stages of the viral life cycle to accomplish the infection. The potential of HSP70s-based antiviral therapeutics is also discussed.
Emergence and geographical extension of dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses increase simultaneous outbreak in an increasing number of countries. To date, no vaccine or cure have yet been developed against these diseases those cause a tremendous impact on human health and in the economy worldwide. During recent simultaneous outbreaks, up to 12% of patients have been diagnosed to be co-infected by CHIKV and DENV. In addition, it was shown that the mosquitoes Aedes albopictus could carry and transmit simultaneously CHIKV and DENV. However, the pathology, as well as the epidemiology of a pathogen, relies on the interactions between several infectious agents present within an organism or a community in the environment. It is crucial to consider to which extent a host infected by a first microorganism is modified and whether its reaction to the infection by a second microorganism is consequently altered. However, there is no extensive report of Alphavirus-Flavivirus or Flavivirus- Flavivirus interactions. Our global objective is to characterize these co-infections in both mosquitoes and humans, at the cell and molecular level. To this aim, we started this project by performing sequential co- infection in different cell lines from Aedes albopictus and Aedes aegypti. We found that the permissiveness and production of DENV is enhanced in presence of CHIKV. On the contrary, there is no effect of DENV pre-infection on subsequent CHIKV co-infection. We generalized the synergistic phenomena and we showed that CHIKV pre-infection also increased the infection by DENV-1, DENV-3 and DENV-4, but also by two others re-emerging Flaviviruses, the Yellow Fever Virus (YFV), and the Zika Virus (ZIKV). Remarkably, we succeeded to establish a mosquito model of co-infection of Aedes aegypti mosquito after by different two feedings at 4 days interval. Using this sequential co-infection, we were able to show that a pre-infection of Aedes aegypti by CHIKV increase the level of DENV-2 RNA in salivary glands compare to mono-infected mosquitos. This phenotype is reminiscent of the phenotype we observed in vitro during successive infections. Altogether, our study paves the way to the characterization of molecular interaction between Flaviviruses and Alphaviruses in mosquito in vitro and in vivo. This study can be crucial for a better understanding of disease and epidemiology during simultaneous outbreaks
Yellow fever virus (YFV) replication is highly dependent upon host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here, interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1- DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein which has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. Fifty-nine proteins were found to be significantly increased when compared to a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher’s exact test was significant for 58 proteins, and the p-value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified sixteen pathways, and this analysis indicated Sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using Sirolimus and Cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.
Phytoplasmas are plant pathogenic, phloem colonizing, cell-wall-less microorganisms that primarily dependent on insect transmission for their spread and survival. The life cycle of phytoplasmas involves replication in insects and host plants. Until recently, phytoplasmas have resisted all attempts at cultivation in cell-free media, making these pathogens poorly characterized on a physiological and biochemical basis. However, host-pathogen relationships can be studied by investigating immunodominant membrane proteins (IDPs), which are located on the exterior surfaces of phytoplasma cells and are the most abundant proteins of the cell membrane. These membrane proteins come in direct contact with both insect and plant hosts and are thought to play a crucial role in phytoplasma spread both within the plant and by insect vectors. Therefore, there is great interest in studying this class of proteins. We summarize and discuss important investigations about these membrane proteins, which have already provided a better understanding of the host-phytoplasma relationship.
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During the infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the involvement of the 70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.
Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase β subunit (ATPSβ). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPSβ. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPSβ showed a significant reduction in viral entry and virus production. These results suggest that ATPSβ is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells.
E2F transcription factor is subject to stringent regulation by a variety of molecules. We recently observed that prohibitin,
a potential tumor suppressor protein, binds to the retinoblastoma (Rb) protein and represses E2F transcriptional activity.
Here we demonstrate that prohibitin requires the marked box region of E2F for repression; further, prohibitin can effectively
inhibit colony formation induced by overexpression of E2F1 in T47D cells. Prohibitin was also found to interact with the signaling
kinase c-Raf-1, and Raf-1 could effectively reverse prohibitin-mediated repression of E2F activity. Agents such as E1A, p38
kinase, and cyclins D and E had no effect on prohibitin-mediated repression of E2F1, but all of these molecules could reverse
Rb function. Similarly, stimulation of the immunoglobulin M signaling pathway in Ramos cells could inactivate prohibitin,
but this had no effect on Rb function. Serum stimulation of quiescent Ramos cells inactivated Rb and prohibitin with different
kinetics; further, while the serum-dependent inactivation of Rb was dependent on cyclin-dependent kinase activity, the inactivation
of prohibitin was not. We believe that prohibitin is a novel regulator of E2F function which channels specific signaling cascades
to the cell cycle regulatory machinery.
Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score >or= +/-2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.
Dengue virus (DENV), the etiological agent of dengue fever, is transmitted to the human host during blood uptake by an infective mosquito. Infection of vector salivary glands and further injection of infectious saliva into the human host are key events of the DENV transmission cycle. However, the molecular mechanisms of DENV entry into the mosquito salivary glands have not been clearly identified. Otherwise, although it was demonstrated for other vector-transmitted pathogens that insect salivary components may interact with host immune agents and impact the establishment of infection, the role of mosquito saliva on DENV infection in human has been only poorly documented. To identify salivary gland molecules which might interact with DENV at these key steps of transmission cycle, we investigated the presence of proteins able to bind DENV in salivary gland extracts (SGE) from two mosquito species. Using virus overlay protein binding assay, we detected several proteins able to bind DENV in SGE from Aedes aegypti (L.) and Aedes polynesiensis (Marks). The present findings pave the way for the identification of proteins mediating DENV attachment or entry into mosquito salivary glands, and of saliva-secreted proteins those might be bound to the virus at the earliest step of human infection. The present findings might contribute to the identification of new targets for anti-dengue strategies.
Tubulin acts as a positive transcription factor for in vitro RNA synthesis by two different negative-strand viruses: Sendai virus, a paramyxovirus; vesicular stomatitis virus (VSV), a rhabdovirus. A monoclonal antibody directed against beta-tubulin completely inhibited not only mRNA synthesis and RNA replication catalyzed in vitro by extracts of cells infected with either virus but also mRNA synthesis by detergent-disrupted purified virions. The synthesis of both a leader-like RNA and the NP mRNA directed by detergent-disrupted purified Sendai virions was shown to be totally dependent on the addition of purified tubulin. The addition of purified tubulin, although not required, also stimulated mRNA synthesis directed by detergent-disrupted VSV virions 2- to 7-fold. Finally, there appears to be an association between tubulin and the L protein of VSV, since both monoclonal and polyclonal anti-tubulin antisera specifically immunoprecipitated not only tubulin but also the L protein of two different VSV serotypes from the soluble protein fraction of infected cells.
Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 micrometer/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 micrometer/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.
Heat shock proteins are molecular chaperones that participate in different cellular processes, particularly the folding and translocation of polypeptides across membranes. In this regard, members of the Hsp70 family of heat shock proteins have been observed in close proximity to cellular membranes. In this study, the direct interaction between Hsc70, which is constitutively expressed in cells, and lipid membranes was investigated. Recombinant Hsc70 was incorporated into artificial lipid bilayers, and a transmembrane ion flow was detected, suggesting the incorporation of an ion pathway. This ion flow was very stable and occurred in well defined, multilevel discrete electrical current events, indicating the formation of a multiconductance ion channel. The Hsc70 channel activity is ATP-dependent and is reversibly blocked by ADP. This channel has cationic selectivity. Thus, Hsc70 can directly interact with lipid membranes to create functionally stable ATP-dependent cationic pathways.
Membrane trafficking from the endoplasmic reticulum (ER) to the Golgi complex is mediated by pleiomorphic carrier vesicles that are driven along microtubule tracks by the action of motor proteins. Here we describe how NSP4, a rotavirus membrane glycoprotein, binds to microtubules and blocks ER-to-Golgi trafficking in vivo. NSP4 accumulates in a post-ER, microtubule-associated membrane compartment and prevents targeting of vesicular stomatitis virus glycoprotein (VSV-G) at a pre-Golgi step. NSP4 also redistributes beta-COP and ERGIC53, markers of a vesicular compartment that dynamically cycles between the ER and Golgi, to structures aligned along linear tracks radiating throughout the cytoplasm. This block in membrane trafficking is released when microtubules are depolymerized with nocodazole, indicating that vesicles containing NSP4 are tethered to the microtubule cytoskeleton. Disruption of microtubule-mediated membrane transport by a viral glycoprotein may represent a novel pathogenic mechanism and provides a new experimental tool for the dissection of early steps in exocytic transport.
The host cytoskeleton plays important roles in the entry, replication, and egress of viruses. An assortment of viruses hijack cellular motor proteins to move on microtubules toward the cell interior during the entry process; others reverse this transport during egress to move assembling virus particles toward the plasma membrane. Polymerization of actin filaments is sometimes used to propel viruses from cell to cell, while many viruses induce the destruction of select cytoskeletal filaments apparently to effect efficient egress. Indeed, the tactics used by any given virus to achieve its infectious life cycle are certain to involve multiple cytoskeletal interactions. Understanding these interactions, and their orchestration during viral infections, is providing unexpected insights into basic virology, viral pathogenesis, and the biology of the cytoskeleton.
We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-beta1 in TF-1 cells.
Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a beta-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.
Binding of dengue virus 2 (DENV-2) to C6/36 mosquito cells protein was investigated. A 48 kDa DENV-2-binding C6/36 cells protein (D2BP) was detected in a virus overlay protein-binding assay. The binding occurred only to the C6/36 cells cytosolic protein fraction and it was inhibited by free D2BP. D2BP was shown to bind to DENV-2 E in the far-Western-binding studies and using mass spectrometry (MS) and MS/MS, peptide masses of the D2BP that matched to beta-tubulin and alpha-tubulin chains were identified. These findings suggest that DENV-2 through DENV-2 E binds directly to a 48 kDa tubulin or tubulin-like protein of C6/36 mosquito cells.
Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-beta-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.
Dengue viruses (DENV) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies.
(1) Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited cell binding and infection. (3) Both proteins were bound by all four serotypes of dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut.
Our results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells.
Japanese encephalitis virus (JEV) is a member of mosquito-borne Flaviviridae. To date, the mechanisms of the early events of JEV infection remain poorly understood, and the cellular receptors are unidentified. There are evidences that the structure of the virus attachment proteins (VAP), envelope glycoprotein of mosquito-borne flaviviruses is very similar, and the vector-virus interaction of mosquito-borne flaviviruses is also very similar. Based on the studies previously demonstrated that the similar molecules present on the mosquito cells involved in the uptake process of JEV, West Nile virus (WNV) and Dengue virus (DV), it is proposed that the same receptor molecules for mosquito-borne flaviviruses (JEV, WNV and DV) may present on the surface of C6/36 mosquito cells. By co-immunoprecipitation assay, we investigated a 74-KDa protein on the C6/36 cells binds JEV, and the mass spectrometry results indicated it may be heat shock cognate protein 70(HSC70) from Aedes aegypti. Based upon some other viruses use of heat shock protein 70 (HSP70) family proteins as cell receptors, its possible HSC70's involvement in the fusion of the JEV E protein with the C6/36 cells membrane, and known form of cation channels in the interaction of HSC70 with the lipid bilayer, it will further be proposed that HSC70 as a penetration receptor mediates JEV entry into C6/36 cells.
Prohibitin, a potential tumor suppressor protein, has been shown to inhibit cell proliferation and repress E2F transcriptional
activity. Though prohibitin has potent transcriptional functions in the nucleus, a mitochondrial role for prohibitin has also
been proposed. Here we show that prohibitin is predominantly nuclear in two breast cancer cell lines where it co-localizes
with E2F1 and p53. Upon apoptotic stimulation by camptothecin, prohibitin is exported to perinuclear regions where it localizes
to mitochondria. The data presented here also show that prohibitin is capable of physically interacting with p53 in vivo and in vitro. Prohibitin was found to enhance p53-mediated transcriptional activity and cotransfection of an antisense prohibitin construct
reduces p53-mediated transcriptional activation. Prohibitin appears to induce p53-mediated transcription by enhancing its
recruitment to promoters, as detected by chromatin immunoprecipitation assays. These results suggest that prohibitin is capable
of modulating Rb/E2F as well as p53 regulatory pathways.
Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells.
(1) We determined the MG proteins that bind DENV by an overlay protein binding assay (VOPBA) in Ae. aegypti mosquitoes of the DS3, DMEB and IBO-11 strains. The main protein identified had an apparent molecular weight of 67 kDa, although the protein identified in the IBO-11 strain showed a lower mass (64 kDa). (2) The midgut proteins recognized by DENV were also determined by VOPBA after two-dimensional gel electrophoresis. (3) To determine whether the same proteins were identified in all three strains, we obtained polyclonal antibodies against R67 and R64 and tested them against the three strains by immunoblotting; both antibodies recognized the 67 and 64 kDa proteins, corroborating the VOPBA results. (4) Specific antibodies against both proteins were used for immunofluorescent location by confocal microscopy; the antibodies recognized the basal lamina all along the MG, and cell membranes and intercellular spaces from the middle to the end of the posterior midgut (pPMG) in the neighborhood of the hindgut. (5) Quantitative analysis showed more intense fluorescence in DS3 and DMEB than in IBO-11. (6) The viral envelope antigen was not homogeneously distributed during MG infection but correlated with MG density and the distribution of R67/R64.
In this paper we provide evidence that the 67 kDa protein (R67/R64), described previously as a DENV receptor, is related to vector competence in Ae. aegypti. Consequently, our results strongly suggest that this protein may be a marker of vector competence for DENV in Ae. aegypti mosquitoes.
Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca2+ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.
Previous studies have shown that West Nile (Sarafend) virus matured by budding at the plasma membrane, which differs from the usual intracellular maturation of other flaviviruses. The present study investigated the trafficking mechanism of the envelope (E) and capsid (C) proteins of West Nile (Sarafend) virus during the replication cycle. The use of time-based double-immunofluorescence labelling coupled with the Triton X-100 extraction procedure revealed that both the E and C proteins were transported from the perinuclear region towards the plasma membrane along the microtubules simultaneously. The strong association of these virus proteins with the microtubules was demonstrated further with Triton X-100 extraction procedure coupled with double immunogold-labelling. Extraction of infected cells with Triton X-100 in high salt also revealed that virus E proteins were associated with the microtubules via protein-protein interaction. The disruption of microtubules with vinblastine sulphate inhibited the trafficking of both the virus E and C proteins. Both virus structural proteins were observed to co-localise and retained within vinblastine sulphate-induced microtubulin paracrystals. Extracellular virus production was also reduced drastically by vinblastine sulphate at non-cytotoxic concentration. Subsequent studies revealed that the transportation of virus E protein was associated with the microtubules-based motor protein, kinesin. J. Med. Virol. 67:127–136, 2002. © 2002 Wiley-Liss, Inc.
Induction of microtubulin paracrystals (Pc) by Kunjin (KUN) virus occurred after 15 hours post-infection and were often associated with convoluted membranes (cm) and virus particles. Vinblastine sulphate which disrupts microtubulin, had an inhibitory effect on the virus production when added during the viral latent period. When infected samples were extracted with Triton X-100 and analysed by SDS-PAGE, four viral proteins were observed.
1.1. Brush border membrane vesicles (BBMV) were prepared from midguts of Pieris brassicae larvae by Mg/EGTA precipitation and differential centrifugation.2.2. Their morphology, polypeptide composition, and marker enzyme enrichment was similar to BBMV from other larval lepidopteran midguts.3.3. In the presence of an inwardly directed KSCN gradient, these BBMV transiently accumulated alanine, phenylalanine, histidine, lysine, or gultamic acid.4.4. Initial uptake rates for neutral and basic amino acids were similar in the presence of either a potassium or a sodium gradient.5.5. Initial uptake of glutamic acid was much more efficient in the presence of a sodium gradient.
Heat shock proteins (Hsps) or stress proteins are highly conserved molecules and expressed in all cell types under stressful conditions like heat, cold, hypoxia and infections. The objective of the present study was to determine the effect of dengue virus infection on relative expression of stress proteins and their role in the progression of the infection. As macrophages are the primary host for dengue, human promonocytic myeloblastoma U937 cells were infected with dengue virus type 2 New Guinea C strain for the evaluation of Hsps expression. A significant expression of Hsp60 was observed in virally infected U937 cells as compared to controls. In order to determine the correlation between Hsp60 expression and viral multiplication in infected cells, expression of Hsp60 was down regulated by RNA interference. Viral multiplication was determined by quantification of viral RNA copy number using Real Time PCR and plaque formation assay in cellular supernatants of Hsp60 silenced cells. Intracellular quantification of viral load was also determined by flow cytometry. It was observed that down regulation of Hsp60 in virally infected cells resulted into decrease in viral RNA copy number, plaque forming units and intracellular viral load. At the same time down regulation also resulted in increased IFN-alpha level. These observations suggest that, elevated levels of Hsp60 expression in virally infected cells may help in viral multiplication and could be possible therapeutic targets for the management of dengue virus infection.
The molecules involved in dengue virus entry into human cells are currently unknown. We have previously shown that two surface heat shock proteins (Hsps), Hsp90 and Hsp70 are part of a receptor complex in monocytic cells. In the present report, the effect of heat shock (HS) on dengue virus infection is analyzed. We have documented a more than twofold increase in dengue virus infectivity after HS treatment in monocytic cells U937; this effect correlates mainly with an increase in viral entry due to a major presence of both Hsps on the surface of monocytic cells, particularly in membrane microdomains. Interestingly, since heat shock treatment at 6h post-infection also increased viral yields, it is likely that HS also modulates positively dengue virus replication.
Virus-induced vesicles evolved early in the Kunjin virus replication cycle around 9 to 10 h p.i. just before the end of the latent period in infected Vero cells. About 2 h following the appearance of the vesicles, microtubule paracrystals were also formed. These two virus-induced structures seemed interlinked and have essential roles in Kunjin virus replication. A viral protein NS3 was found to be associated with the microtubule component of the cells. When vinblastine sulphate was added to the cells immediately after infection, formation of the paracrystals was delayed by two hours, and the affiliation of NS3 protein was also observed to be rearranged.
Induction of microtubulin paracrystals (Pc) by Kunjin (KUN) virus occurred after 15 hours post-infection and were often associated with convoluted membranes (cm) and virus particles. Vinblastine sulphate which disrupts microtubulin, had an inhibitory effect on the virus production when added during the viral latent period. When infected samples were extracted with Triton X-100 and analysed by SDS-PAGE, four viral proteins were observed.
Prohibitin is an evolutionarily conserved gene with homologues found in organisms ranging from yeast to man. In man the gene is located on chromosome 17 at q21. The deduced amino acid sequences of the protein products from mouse and rat are identical; and these differ from the human protein sequence by a single conserved amino acid. Prohibitin has antiproliferative activity and available data suggest a role in such diverse processes as normal cell cycle regulation, replicative senescence, cellular immortalization, and the development of sporadic breast tumors. Although its functional activity is presently unknown, the 30,000-Da protein has been located in the inner membrane of mitochondria, where it is postsynthetically modified, as well as on the plasma membrane of B cells, where it is associated with the IgM receptor. Prohibitin's evolutionary conservation and ubiquitous expression indicate that it is a fundamentally important gene; and current data suggest a functional role in such dissimilar processes as development, senescence, and tumor suppression.
Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.
The initial steps of dengue viral entry have been divided into adsorption and penetration using acid glycine treatment to inactivate extracellular virus after attachment to baby hamster kidney (BHK) cells but prior to penetration. First, we showed that virus infection was accomplished within 2 h after adsorption. Second, the assay was used to examine the properties of dengue envelope E protein-specific monoclonal antibodies (MAbs), lectins, and heparin. We found that three MAbs, 17-2, 46-9, and 51-3, may neutralize dengue 2 virus (DEN-2) through inhibition of not only viral attachment but also of penetration. However, one MAb, 56-3.1, interfered specifically with attachment. Therefore, the functional domains of E protein involved in attachment and penetration may be different. Moreover, studies with lectins indicated that carbohydrates, especially alpha-mannose residues, present on the virion glycoproteins may contribute to binding and penetration of the virus into BHK and mosquito C6/36 cells. Finally, virus infectivity was inhibited by heparin through its blocking effects at both virus attachment and penetration. This suggests that cell surface heparan sulfate functions in both viral attachment and penetration of DEN-2 virus. In conclusion, our results further elucidated some aspects of the dengue virus entry process.
Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature (4 degrees C), BPV-1 binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electron microscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymerizing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane.
Varicella-zoster virus (VZV) open reading frame 17 (ORF 17) is the gene corresponding to Herpes simplex-virus (HSV) UL41. The UL41 gene encodes the virion host shutoff factor (vhs), a RNase that has been the object of detailed studies. In contrast to HSV, knowledge about VZV mediated shutoff effects and the role of ORF 17 is poor.
We investigated the ORF 17 expression in infected cells and analyzed shutoff effects. ORF 17 expression could not be proven in infected human fibroblast cell lines and melanoma (MeWo) cells. Only after induction by Phorbol 12-myristate 13-acetate an ORF 17 expression became detectable in MeWo cells. Nevertheless, using stable expressed GAPDH mRNA as a marker for mRNA degradation, a VZV mediated shutoff, independent of ORF 17 expression, became measurable. Transfection experiments demonstrated that transient ORF 17 expression did not decrease the cellular GAPDH mRNA level. We examined whether the VZV shutoff factor is a tegument protein causing an early shutoff or whether it needs to be expressed (delayed shutoff). The GAPDH mRNA level in Actinomycin D pretreated and infected MeWo cells did not decrease even faster than the theoretical decay rate based on a half-life of 24 h. These findings lead to the conclusion that the VZV shutoff factor is not a mature protein localized in the virion and that VZV causes a delayed virion host shutoff effect.
Aedes aegypti is the primary vector of yellow fever (YF) and dengue fever (DF) flaviviruses worldwide. In this review we focus on past and present research on genetic components and environmental factors in Aedes aegypti that appear to control flavivirus transmission. We review genetic relationships among Ae. aegypti populations throughout the world and discuss how variation in vector competence is correlated with overall genetic differences among populations. We describe current research into how genetic and environmental factors jointly affect distribution of vector competence in natural populations. Based on this information, we propose a population genetic model for vector competence and discuss our recent progress in testing this model. We end with a discussion of approaches being taken to identify the genes that may control flavivirus susceptibility in Ae. aegypti.
Dengue virus (DENV) infects target cells by attaching to various cell receptors, many of which are still unknown. In C6/36 cells (Aedes albopictus cell line), DENV-4 bound to two glycoproteins of 40 and 45 kDa, located on the cell surface. Preincubation of cells with polyclonal antibody against the 45-kDa protein specifically blocked DENV-4 infection of C6/36 cells. The antibody and purified DENV-4 detected the 45-kDa molecule in total extracts from eggs, larvae, and pupae as well as from the midgut, ovary, and salivary glands from adult-stage Aedes aegypti mosquitoes, whereas in malphigian tubules it was absent. This suggests that the distribution of the 45-kDa protein correlates with tissue tropism of DENV infection in mosquitoes. The 45-kDa molecule was not detected in Anopheles albimanus mosquito. The relevance of our findings is discussed from the pathogenetic and vector competence viewpoints.
Flaviviruses are spherical enveloped viruses with a diameter of approximately 50 nm that contain only three structural proteins: E (envelope), prM/M (membrane), and C (capsid). This chapter focuses primarily on structural aspects of the envelope glycoproteins, their organization in the viral envelope, and the mechanism of virus-induced membrane fusion. Significant progress has been made toward the understanding of the structure and organization of the flavivirus virion. The current view of the flavivirus life cycle, including entry, assembly, maturation, and release suggests that virus entry occurs by receptor-mediated endocytosis, and the acidic pH in the endosome triggers structural alterations in the E protein that lead to the fusion of the viral membrane with the endosomal membrane and the release of the nucleocapsid. During assembly, immature prM-containing virions are believed to be formed in the endoplasmic reticulum (ER) and transported through the secretory pathway of the cell. The most important structural features revealed by current studies are: an icosahedral arrangement of the envelope proteins and a characteristic envelope protein structure, which, together with the alphavirus E1 protein, defines a separate class of viral fusion protein.
Aedes aegypti was eliminated from Brazil in 1955, but re-infested the country in the 1970s. Dengue outbreaks have occurred since 1981 and
became endemic in several cities in Brazil after 1986. Urban yellow fever has not occurred since 1942, and only jungle yellow
fever cases have been reported. A population genetic analysis using isoenzyme variation combined with an evaluation of susceptibility
to both yellow fever and dengue 2 viruses was conducted among 23 A. aegypti samples from 13 Brazilian states. We demonstrated that experimental infection rates of A. aegypti for both dengue and yellow fever viruses (YFV) are high and heterogeneous, and samples collected in the endemic and transition
areas of sylvatic yellow fever were highly susceptible to yellow fever virus. Boa Vista, a border city between Brazil and
Venezuela, and Rio de Janeiro in the Southeast region are considered as the most important entry points for dengue dissemination.
Considering the high densities of A. aegypti, and its high susceptibility to dengue and yellow fever viruses, the risk of dengue epidemics and yellow fever urbanization
in Brazil is more real than ever.
Permeability alterations of microvascular endothelia may be a factor in the plasma leakage produced by dengue virus infection. Confluent monolayers of the human dermal microvascular endothelial cell line HMEC-1 were utilized as an experimental model to study the cellular responses induced by the virus. Infected monolayers showed increased permeability for [(3)H]mannitol, but no changes were observed for 4-70 kDa dextrans at 48 h post-infection (p.i.), a time at which viral titres reached maximal values and 40 % of the cells expressed viral proteins. A further increase in permeability occurred at 72 h, still without evident cytopathic effects on the monolayer. Coinciding with this, actin was reorganized in the infected cells and the tight junction protein occludin was displaced to the cytoplasm. Increments in the thickness of stress fibres and focal adhesions were observed in uninfected cells neighbouring infected cells. Culture medium from infected monolayers induced permeability changes and thickening of actin-containing structures in control cultures that resembled those observed 48 h p.i. Interleukin (IL) 8 was found in culture medium at concentrations ranging from 20 to 100 pg ml(-1). Neutralizing antibodies against IL8 partially inhibited the changes produced by the culture medium as well as those induced by addition of IL8. Genistein inhibited the effect of the culture medium and the phosphorylation of proteins associated with focal adhesions and indicated the participation of tyrosine kinases. These findings suggest that IL8 production by infected monolayers contributes to the virus-induced effect on the cytoskeleton and tight junctions and thereby modifies transendothelial permeability.
Vi capsular polysaccharide (Vi) was first identified as a virulence antigen of Salmonella typhi, the causative agent of typhoid fever in humans; it renders S. typhi resistant to phagocytosis and the action of serum complement. However, the role of Vi during the infection of intestinal epithelium with S. typhi is not completely understood. We show here that Vi can interact with a model human intestinal epithelial cell line, Caco-2, through a cell-surface-associated molecular complex containing two major proteins of 30 and 35 kDa and a minor protein of ≈68 kDa. The two major proteins were identified as the putative tumor suppressor molecule, prohibitin, and its closely related homolog, B cell receptor-associated protein 37. These two proteins were enriched in lipid rafts, and Vi readily associated with these membrane microdomains. Engagement of Caco-2 cells with Vi inhibited their ability to produce an inflammatory response upon infection with Vi–S. typhi. Consistent with this effect, infection of Caco-2 cells with Vi⁺S. typhi produced less IL-8 compared with Vi–S. typhi. Cells treated with Vi showed reduced extracellular signal-regulated kinase phosphorylation in response to infection with Vi–S. typhi or stimulation with phorbol 12-myristate 13-acetate, suggesting that the mitogen-activated protein kinase pathway might be a target for Vi-mediated inhibition of inflammatory responses. These findings reveal a crucial role for Vi in the modulation of early inflammatory responses during infection with S. typhi. This kind of a modulation could play a significant role in the establishment of infection by S. typhi.
• Vi capsular polysaccharide
• putative tumor suppressor molecule
• IL-8
The prohibitins, Phb1 and Phb2 are highly conserved proteins in eukaryotic cells that are present in multiple cellular compartments. Initial investigations focused on the role of Phb1 as an inhibitor of cell proliferation hence the original name prohibitin. However both proteins appear to have a diverse range of functions and recent evidence suggests that the prohibitins have very similar but as yet only partially understood functions. In addition to their role as chaperone proteins in the mitochondria, and their ability to target to lipid rafts, their is now compelling evidence that both prohibitins are localized in the nucleus and can modulate transcriptional activity by interacting with various transcription factors, including the steroid hormone receptors, either directly or indirectly. In addition Phb1 and Phb2 are present in the circulation and can be internalized when added to cultured cells suggesting that the circulating prohibitins may have some regulatory role. This review presents some of the recent developments in prohibitin research and focuses on the similarities in the structure and function of these interesting proteins.
Prohibitin (PHB1) is a multifunction protein that is released in lipid droplets from adipocytes and possibly other cells and is detectable in the circulation. We used crosslinking, immunoprecipitation and proteomic analysis to investigate binding partners for circulating PHB1. Crosslinking of PHB1 to serum resulted in two complexes of approximately 150 and 100 kDa, which contained both PHB1 and fragments of C3. The binding of PHB1 to C3 was confirmed using a solid phase assay where the dissociation constant was approximately 90 fmol/l. PHB1, but not the closely related PHB2, was able to enhance complement activation and induce lysis of sensitized sheep erythrocytes when added with normal serum but not with C3-deficient serum. The ability of PHB1 to bind to, and activate C3 suggests that PHB1 may have a previously unrecognized role in innate immunity.
Dengue virus (DENV) is transmitted to humans by mosquitoes of the genus Aedes. Although several molecules have been described as part of DENV receptor complex in mosquito cells, none of them have been identified. Our group characterized two glycoproteins (40 and 45 kD) as part of the DENV receptor complex in C6/36 cells. Because identification of the mosquito cell receptor has been unsuccessful and some cell receptors described for DENV in mammalian cells are heat-shock proteins (HSPs), the role of HSPs in DENV binding and infection in C6/36 cells was evaluated. Our results indicate that gp45 and a 74-kD molecule (p74), which interact with DENV envelope protein, are immunologically related to HSP90. Although p74 is induced by heat shock, gp45 apparently is not. However, these proteins are relocated to the cell surface after heat-shock treatment, causing an increase in virus binding without any effect on virus yield.
Several studies have identified putative dengue virus receptors using virus overlay protein binding assays (VOPBA) with some apparent success. Given that this technique relies upon the use of electrophoresis of proteins through polyacrylamide gels with varying amounts of protein denaturation, the physiological relevance of the proteins isolated is open to question. To address this issue a Sepharose 4B-dengue virus serotype 2-affinity column was constructed to selectively bind dengue virus binding proteins from HepG2 (liver) cell membrane preparations. Results show that GRP78, but not the 37/67 kDa high affinity laminin receptor, was specifically bound by the column. This result is consistent with earlier work and shows that while affinity chromatography may provide a useful adjunct to VOPBA based studies particularly in cases where proteins maybe sensitive to denaturation, proteins isolated by VOPBA can be physiologically relevant.
Identification of dengue virus binding proteins using affinity chromatography Villas Dengue virus-induced regulation of the host cell translational machinery
- S Upanan
- A Kuadkitkan
- Smith
- Csa
- Tm Conceição
- J Ramírez
- Da Abm Santoro
- At Poian
- Montero
- Lomelí
Upanan S, Kuadkitkan A, Smith DR (2008) Identification of dengue virus binding proteins using affinity chromatography. J Virol Methods 151:325–328 1460 M. S. Paingankar et al. 123 35. Villas-Bôas CSA, Conceição TM, Ramírez J, Santoro ABM, Da Poian AT, Montero-Lomelí M (2009) Dengue virus-induced regulation of the host cell translational machinery. Braz J Med Biol Res 42(11):1020–1026