FIG 1 - uploaded by Sheila M Nolan
Content may be subject to copyright.
Generation and replication of cDNA-derived HPIV-2 with mutations in V only. (A) Schematic representation of the rHPIV-2-WT genome. The nucleotide sequence at the P/V mRNA editing site is shown for the unedited mRNA encoding V and the edited mRNA encoding P, with underlining denoting the seven (V mRNA) or nine (P mRNA) G residues. Codons 164 and 165 in each mRNA, marking the junction at which editing changes the reading frame, are boxed. Diagrams of the complete V and P proteins are shown to the right; as indicated, P and V share identical N-terminal sequences (164 amino acids in length) but have unique C-terminal sequences after the editing site. (B) Diagram of the rHPIV-2-P ϩ V genome with separated P and V genes, which was used as the backbone to create the mutants described in the present study. The unique restriction enzyme sites used to insert mutated V open reading frames (ORFs) are indicated. (C) Using the HPIV-2-P ϩ V cDNA, mutations were introduced separately into the C-terminal domain (CTD) of the HPIV-2 V protein encoded by the separated V-only gene unit, without affecting P. (D) Representation of the CTD zinc-finger fold predicted from the crystal structure of the PIV5 V protein (30). Two loops coordinate two zinc ions via amino acids H174 (H), C193 (C1), C197 (C2), C209 (C3), C211 (C4), C214 (C5), C218 (C6), and C221 (C7). (E) Multicycle replication in LLC-MK2 cells infected at an MOI of 0.01 TCID 50 /cell. Replication studies were performed in triplicate, and mean titers for each time point are indicated.
Source publication
Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 2...
Contexts in source publication
Context 1
... were introduced into the V gene by two-step PCR mutagenesis using the Advantage-HF PCR kit (Clontech Laboratories, Palo Alto, CA) and mutagenic primers. The mutated V gene was cloned into the AgeI and SacII sites of the rHPIV-2-PV antigenomic sense cDNA (Fig. 1B). All regions of the mutated cDNAs that had been subjected to in vitro polymerase reactions were sequenced using a Perkin-Elmer ABI 3730 sequencer with the BigDye sequenc- ing kit version 1.1 (Perkin-Elmer Applied Biosystems, Warrington, United King- dom) as previously described ...
Context 2
... are highly conserved among all paramyxovirus V proteins: six in- dividual cysteine-to-serine substitutions (residues 193, 197, 209, 211, 214, and 218, corresponding to C1 to C6 in the conserved motif), one histidine-to-phenylalanine substitution (H174F), and two paired charge-to-alanine mutations (R175A/ R176A and R205A/K206A), as indicated in Fig. 1C. Their positions in a proposed zinc-binding domain based on PIV5 (29) are shown in Fig. 1D. Codons specifying the intended substitutions were chosen so that each differed by as many nucleotides as possible from the codon encoding the respective WT assignment. This was done to reduce the frequency of same-site reversion and to increase ...
Context 3
... substitutions (residues 193, 197, 209, 211, 214, and 218, corresponding to C1 to C6 in the conserved motif), one histidine-to-phenylalanine substitution (H174F), and two paired charge-to-alanine mutations (R175A/ R176A and R205A/K206A), as indicated in Fig. 1C. Their positions in a proposed zinc-binding domain based on PIV5 (29) are shown in Fig. 1D. Codons specifying the intended substitutions were chosen so that each differed by as many nucleotides as possible from the codon encoding the respective WT assignment. This was done to reduce the frequency of same-site reversion and to increase the genetic stability of each mutation so that it could be used as an attenuating mutation ...
Context 4
... be used as an attenuating mutation in a live attenuated HPIV-2 vaccine virus (32). Sequences for the original and mutant codons are shown in Table 1. These mu- tations were introduced into a previously described version of the HPIV-2 antigenomic cDNA (HPIV-2-PV) in which the P and V coding regions were engineered to be expressed as separate mRNAs (Fig. 1B); this was done to permit stabiliza- tion of the V mutations without altering the coding sequence of the phosphoprotein P. From the nine mutated cDNAs, six mutant viruses could be recovered (Table 1). Viruses contain- ing any of the six cysteine substitutions were difficult to recover from cDNA, yielding only three viable viruses (the ...
Context 5
... was assessed in LLC-MK2 cells, in which HPIV-2-WT replicates very efficiently. Upon infection at a low MOI (0.01 TCID 50 /cell), multicycle replication of the C218S mutant was below the limit of detection (data not shown), while replication of the C193S mutant was significantly reduced (1,000-fold-lower peak titer compared to that of rHPIV-2-PV) (Fig. 1E). The poor replication of the C218S and C193S mutants prevented further analysis of these viruses. The remaining mutants replicated to peak titers that were comparable to that of the rHPIV-2-PV parent (Fig. 1E). These four mutants were also evaluated in single-step growth curves (see Fig. 3C), and their replication was equivalent to ...
Context 6
... not shown), while replication of the C193S mutant was significantly reduced (1,000-fold-lower peak titer compared to that of rHPIV-2-PV) (Fig. 1E). The poor replication of the C218S and C193S mutants prevented further analysis of these viruses. The remaining mutants replicated to peak titers that were comparable to that of the rHPIV-2-PV parent (Fig. 1E). These four mutants were also evaluated in single-step growth curves (see Fig. 3C), and their replication was equivalent to that of rHPIV-2-WT and rHPIV-2-PV. None of the V mu- tants examined here induced cell death, in contrast to previous observations with the V knockout mutant rHPIV-2-V ko (data not shown) (53). Elevated ...
Context 7
... are one histidine (H) and seven cysteine (C) residues that form a type of zinc finger resembling a RING domain. For PIV5, structural data indicated that the first histidine (residue H174 in HPIV-2) and C1, -6, and -7 coordinate one zinc atom (loop 1), while C2, -3, -4, and -5 are part of a smaller loop that coordinates a second zinc atom (loop 2) (Fig. 1D) (29). Nishio et al. had previously generated HPIV-2 mutants with Cys-to-Ala substitutions at C1C2, C3C4, and C6 (41). Although these mutants could be recov- ered from cDNA, they were highly restricted in replication in vitro (40,41). In contrast, several of our mutants bearing indi- vidual Cys-to-Ser substitutions could not be ...
Context 8
... these adventitious mutations were compensatory, the difficulty of recovering these mutants suggests that these substitutions conferred a more deleterious phenotype than the previously reported mutations. In our system, individual sub- stitution of C2, -3, and -4 (loop 2) was lethal, and substitution of C1 and C6 (in loop 1 but adjacent to loop 2; Fig. 1D) restricted in vitro replication significantly. One possible expla- nation for the difference in the observed lethality of substitu- tions in loop 2 is that we introduced individual substitutions only, which may be more disruptive structurally than substitu- tions of Cys pairs, as reported by Nishio et al. (41). In this regard, we note ...
Context 9
... study, using measles virus, suggested that the production of IFN-was reduced in human epithelial cells in which either RIG-I or MDA5 had been knocked down, indicating that perhaps both proteins have a role in sensing paramyxoviruses (22). Although the mechanism of paramyxovirus V protein binding to MDA5 is well described (1a, 6), knowledge regarding the in vivo conse- quences of the V-MDA5 interaction has otherwise been very limited thus far. To our knowledge, this is the first report of a V protein-specific phenotype in vivo. ...
Citations
... For example, our data showed that a vANI95 representative, ParaV-10A, was closely related to HPIV-2 and simian parainfluenza virus. HPIV-2 and 4 are the causal agents of respiratory disease and can lead to severe outcomes [67]. Considering the recent identification of a close relative of parainfluenza virus 4 (another virus of human health concern, Orthorubulavirus) in Eptesicus bats [68], bats may harbor the common ancestor of diverse parainfluenza viruses circulating in humans. ...
Background
Zoonotic viruses cause substantial public health and socioeconomic problems worldwide. Understanding how viruses evolve and spread within and among wildlife species is a critical step when aiming for proactive identification of viral threats to prevent future pandemics. Despite the many proposed factors influencing viral diversity, the genomic diversity and structure of viral communities in East Africa are largely unknown.
Results
Using 38.3 Tb of metatranscriptomic data obtained via ultradeep sequencing, we screened vertebrate-associated viromes from 844 bats and 250 rodents from Kenya and Uganda collected from the wild. The 251 vertebrate-associated viral genomes of bats (212) and rodents (39) revealed the vast diversity, host-related variability, and high geographic specificity of viruses in East Africa. Among the surveyed viral families, Coronaviridae and Circoviridae showed low host specificity, high conservation of replication-associated proteins, high divergence among viral entry proteins, and frequent recombination. Despite major dispersal limitations, recurrent mutations, cocirculation, and occasional gene flow contribute to the high local diversity of viral genomes.
Conclusions
The present study not only shows the landscape of bat and rodent viromes in this zoonotic hotspot but also reveals genomic signatures driven by the evolution and dispersal of the viral community, laying solid groundwork for future proactive surveillance of emerging zoonotic pathogens in wildlife.
F1ZzYbc5Jf4XpNJGrZx-82Video Abstract
... Ultimately, V protein was proved to inhibit lysine 63 (K63)-linked polyubiquitination of IRF7 mediated by TRAF6, consequently preventing TLR7/9-dependent IFN production (Kitagawa et al., 2013). Apart from V protein discussed above, the C protein of SeV can bind to IKKα and inhibit the phosphorylation of Andrejeva et al., 2004;Komatsu et al., 2007;Sakaguchi et al., 2011;Schaap-Nutt et al., 2011;Svitek et al., 2014;Audsley et al., 2016;Sanz Bernardo et al., 2017 UBXN1 V (NiV) Uchida et al., 2018 PP1α/γ V (MeV and NiV) Davis et al., 2014 LGP2 V (HeV, HPIV2 Garcin et al., 2002;Nishio et al., 2002;Takeuchi et al., 2003;Shaw et al., 2004;Nishio et al., 2005;Nanda and Baron, 2006;Caignard et al., 2007;Devaux et al., 2007Devaux et al., , 2013Svitek et al., 2014;Ma et al., 2015;Oda et al., 2015;Li P. et al., 2019;Keiffer et al., 2020;Nagano et al., 2020 STAT2 V (CDV, HPIV4, MeV, MuV, NiV, PIV5, and PPRV) Nishio et al., 2002Nishio et al., , 2005Rodriguez et al., 2004;Ramachandran et al., 2008;Svitek et al., 2014;Nagano et IRF7, leading to the inhibition of TLR7/9 signaling. However, compared to full size C protein (aa 1-204), Y1 (aa 24-204), and Y2 (aa 30-204) could bind to IKKα and exhibit less ability to suppress TLR7/9 signaling, indicating that N-terminal of C protein is not required for interaction with IKKα. ...
... Further studies screened 20 mutant V proteins of HPIV2 to identify the key residues involved in MDA5 binding. The region comprised of 175-180 is essential for binding to MDA5 (Schaap-Nutt et al., 2011). Phosphatase PP1, such as PP1α and PP1γ, are key regulators of RIG-I and MDA5 activation. ...
For efficient replication, viruses have developed multiple strategies to evade host antiviral innate immunity. Paramyxoviruses are a large family of enveloped RNA viruses that comprises diverse human and animal pathogens which jeopardize global public health and the economy. The accessory proteins expressed from the P gene by RNA editing or overlapping open reading frames (ORFs) are major viral immune evasion factors antagonizing type I interferon (IFN-I) production and other antiviral innate immune responses. However, the antagonistic mechanisms against antiviral innate immunity by accessory proteins differ among viruses. Here, we summarize the current understandings of immune evasion mechanisms by paramyxovirus accessory proteins, specifically how accessory proteins directly or indirectly target the adaptors in the antiviral innate immune signaling pathway to facilitate virus replication. Additionally, some cellular responses, which are also involved in viral replication, will be briefly summarized.
... Since the mid-1980s, this has been a closed animal breeding colony, with no new animals imported since that time. Previous work on parainfluenza virus infection of AGM (e.g., Schaap-Nutt et al., 2011) showed that animals elicited strong levels of serum antibodies, but the origin of the animals used in these studies was not detailed. Thus, the restricted breeding within a closed colony such as the WFU VRC may contribute to genetic bottlenecks in the ability to generate a range of antibodies that have varying degrees of dependence on C'. ...
... All procedures were approved by the Wake Forest University Animal Care and Use Committee. Procedures were based on published approaches for infection of AGM with HPIV2 (Schaap-Nutt et al., 2011). Healthy 3 year-old African Green Monkeys (Chlorocebus aethiops) were sedated with ketamine, and placed in a dorsal position before receiving 10 8 PFU of purified PIV5-gp160 delivered in a total of 2 ml of sterile PBS. ...
The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens.
... Several species of NHPs have previously been used to study AI virus infection and vaccine efficacy (35)(36)(37)(38). African green monkeys (AGMs) have been successfully used as NHP models for other respiratory viruses, including parainfluenza viruses (39,40), respiratory syncytial virus (41), and severe acute respiratory syndrome (SARS) coronavirus (42). Therefore, we undertook an evaluation of several different subtypes of influenza A viruses in AGMs. ...
Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates.
... Similarly, a human parainfluenza virus type 2 (hPIV2) with V protein mutations that prevented the virus from inhibiting IFN-mediated signaling maintained its capacity to replicate in the respiratory tract of non-human primates [53]. In both cases, the capacity to block IFN-a/b induction through interactions with MDA5 and LGP2 probably compensates to some point for the lack of inhibition downstream of IFN-a/b receptor and the same could be true for TioV in vivo [54]. However, the capacity of Paramyxoviridae to target STAT proteins has also been associated to their replication level in a specific host as aforementioned for PIV5 infection in mice [37,38,39], but also to the development of pathology. ...
The capacity of a virus to cross species barriers is determined by the development of bona fide interactions with cellular components of new hosts, and in particular its ability to block IFN-α/β antiviral signaling. Tioman virus (TioV), a close relative of mumps virus (MuV), has been isolated in giant fruit bats in Southeast Asia. Nipah and Hendra viruses, which are present in the same bat colonies, are highly pathogenic in human. Despite serological evidences of close contacts between TioV and human populations, whether TioV is associated to some human pathology remains undetermined. Here we show that in contrast to the V protein of MuV, the V protein of TioV (TioV-V) hardly interacts with human STAT2, does not degrade STAT1, and cannot block IFN-α/β signaling in human cells. In contrast, TioV-V properly binds to human STAT3 and MDA5, and thus interferes with IL-6 signaling and IFN-β promoter induction in human cells. Because STAT2 binding was previously identified as a host restriction factor for some Paramyxoviridae, we established STAT2 sequence from giant fruit bats, and binding to TioV-V was tested. Surprisingly, TioV-V interaction with STAT2 from giant fruit bats is also extremely weak and barely detectable. Altogether, our observations question the capacity of TioV to appropriately control IFN-α/β signaling in both human and giant fruit bats that are considered as its natural host.
... One of the principal triggers of the cellular innate response to viral infection is the detection of viral nucleic acid by cytosolic pattern recognition receptors such as RIG-I and MDA5, which initiate signaling cascades that lead to the activation of cellular transcription factors and expression of type I IFNs, resulting in the induction of an antiviral state in the infected cell as well as neighboring cells, and expression of pro-inflammatory cytokines (Boonyaratanakornkit et al., 2011;Schaap-Nutt et al., 2011). Sideby-side comparison of cytokine secretion showed that PIV2 induced significant apical and basolateral IFN-a secretion by day 2 pi, while PIV3 took three days to induce basolateral IFNa, and PIV1 did not induce much apical IFN-a at all (Fig. 1B and Fig. 2). ...
... Although viral dsRNA accumulation was not examined here, the lack of type I IFN induction by PIV1 suggests that the underlying mechanism in HAE might be the same (Bartlett et al., 2008). For PIV2, the binding of the viral V protein to MDA5 is known to play a role in inhibiting IFN-b expression (Schaap-Nutt et al., 2011). ...
Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.
... Dans la protéine MDA5, la région ciblée par la protéine V est le domaine hélicase responsable de la détection d'ARNdb si l'on considère que MDA5 n'est pas le senseur majeur des paramyxovirus. Cependant, des études récentes démontrent un rôle important de MDA5 dans le déclenchement in vivo et in vitro de la réponse antivirale contre ces virus [24][25][26]. Par ailleurs, il a récemment été démontré que LGP2 (laboratory of genetics and physiology 2) est une cible cellulaire également partagée par l'ensemble des Paramyxovirinae (MuV, MeV, PIV5, NiV...) [27]. L'interaction V-LGP2 implique les mêmes régions (côté virus et côté cellule) que celles impliquées dans l'interaction V-MDA5 [27]. ...
Host immunity against viruses integrates both innate and adaptive responses. Innate immunity represents the first line of defense triggered by viral infections whereas adaptive immunity allows for the establishment of long-term memory. Initially described for their antiviral activity, type I interferons are also involved in the induction of all immune responses. Since these cytokines are crucial for the establishment of an efficient antiviral response, viruses have developped mechanisms to block their expression and signals. The family of Paramyxoviridae presents a wonderful example of how viruses escape the antiviral response. Evasion strategies mainly involve the non structural proteins encoded by the gene of the phosphoprotein (P). These virulence factors can simultaneously or sequentially interact with cellular proteins through multiple interfaces and sophisticated mechanisms. This review describes the different mechanisms developped by paramyxoviruses to hijack the type I IFN response and molecular details, in particular protein-protein interactions, will be highlighted.
... HPIV2 V protein inhibits IFN signaling by binding to and promoting the degradation of STAT2 [49,50]. Overall, inhibition of IFN induction appears to be more important for efficient HPIV2 replication in vivo than inhibition of IFN signaling: recombinant HPIV2 viruses carrying V protein point mutations allowing IFN induction are attenuated in non-human primates [51], while HPIV2 viruses carrying V protein point mutations allowing IFN signaling are not attenuated [44]. This difference may reflect the ability of secreted IFN to affect cells beyond those directly infected with virus, while virusmediated inhibition of IFN signaling operates only in infected cells. ...
Human parainfluenza viruses (HPIVs) are a common cause of acute respiratory illness throughout life. Infants, children, and the immunocompromised are the most likely to develop severe disease. HPIV1 and HPIV2 are best known to cause croup while HPIV3 is a common cause of bronchiolitis and pneumonia. HPIVs replicate productively in respiratory epithelial cells and do not spread systemically unless the host is severely immunocompromised. Molecular studies have delineated how HPIVs evade and block cellular innate immune responses to permit efficient replication, local spread, and host-to-host transmission. Studies using ex vivo human airway epithelium have focused on virus tropism, cellular pathology and the epithelial inflammatory response, elucidating how events early in infection shape the adaptive immune response and disease outcome.
... Measles virus and other morbillivirus V and P proteins bind to STAT1 via a conserved tyrosine motif present on their shared N-terminal domain (61), away from the Jak1 binding site. The unique C-terminal domain of V binds to STAT2 via two conserved tryptophan residues (35) that are located away from the MDA5 binding site (227,248). Together with the ability of V protein to form macromolecular complexes including STAT1, STAT2, and IRF9 (210), this suggests that V protein acts as a molecular scaffold, which prevents both STAT1/2 phosphorylation (275) and nuclear translocation of the ISGF3 complex. A single VOL. ...
The discovery of a new class of cytosolic receptors recognizing viral RNA, called the RIG-like receptors (RLRs), has revolutionized our understanding of the interplay between viruses and host cells. A tremendous amount of work has been accumulating to decipher the RNA moieties required for an RLR agonist, the signal transduction pathway leading to activation of the innate immunity orchestrated by type I interferon (IFN), the cellular and viral regulators of this pathway, and the viral inhibitors of the innate immune response. Previous reviews have focused on the RLR signaling pathway and on the negative regulation of the interferon response by viral proteins. The focus of this review is to put this knowledge in the context of the virus replication cycle within a cell. Likewise, there has been an expansion of knowledge about the role of innate immunity in the pathophysiology of viral infection. As a consequence, some discrepancies have arisen between the current models of cell-intrinsic innate immunity and current knowledge of virus biology. This holds particularly true for the nonsegmented negative-strand viruses (Mononegavirales), which paradoxically have been largely used to build presently available models. The aim of this review is to bridge the gap between the virology and innate immunity to favor the rational building of a relevant model(s) describing the interplay between Mononegavirales and the innate immune system.
... 66,67 Fifth, mutations that inactivate viral interferon antagonists can lead to increased type 1 interferon production in the host and thereby increase vaccine immunogenicity. [68][69][70][71][72] ...
Viral respiratory infections cause significant morbidity and mortality in infants and young children as well as in at-risk adults and the elderly. Although many viral pathogens are capable of causing respiratory disease, vaccine development has to focus on a limited number of pathogens, such as those that commonly cause serious lower respiratory illness (LRI). Whereas influenza virus vaccines have been available for some time (see the review by Clark and Lynch in this issue), vaccines against other medically important viruses such as respiratory syncytial virus (RSV), the parainfluenza viruses (PIVs), and metapneumovirus (MPVs) are not available. This review aims to provide a brief update on investigational vaccines against RSV, the PIVs, and MPV that have been evaluated in clinical trials or are currently in clinical development.
