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Location of the RNA-protein cross-link site. Partial digests of 5'-end-labeled RNA were obtained with RNases T1 (lanes G) and U2 (lanes A). OH, partial alkaline degradation; und., undigested RNA; cont., partial alkaline degradation of a 30-nucleotide-long synthetic RNA (U.S. Biochemical).
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Interaction between viral proteins and RNAs has been studied in rotavirus-infected cells. The use of UV cross-linking followed by immunoprecipitation and labeling with T4 polynucleotide kinase allowed us to detect interactions between RNA and nonstructural viral proteins. The RNAs linked to the nonstructural protein NSP3 have been identified as rot...
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... common characteristic of the eight RNAs was that upon partial alkaline degradation, the penultimate fragment mi- grated two nucleotides shorter than the full-length fragment and one nucleotide longer than the largest fragment obtained with RNase U2 (Fig. 9). Similar gaps have been previously observed with cross-linked material and are attributable to the amino acid covalently linked to the nucleic acid under UV irradiation (4). The position of this gap clearly estab- lished that NSP3 was cross-linked to the last nucleotide of the RNA and that one or two amino acids were that the ...
Citations
... Each dsRNA segment serves as a template for transcribing differently sized (0.67-3.4 kb) protein-coding + ssRNA transcripts (mRNAs) that can fold into regulatory structures, such as those predicted to be found in the 5 and 3 untranslated regions (UTRs) (4,5). In addition, consensus sequences found in 5 and 3 terminal regions of these segments also have important regulatory roles in ensuring proper viral infection, such as recruiting viral proteins to enhance viral translation (6,7). Li et al. provided one of the most comprehensive studies of rotavirus RNAs based on the minimum free energy consensus structures deduced from multiple sequence alignments and covariation analysis of group A rotavirus (RVA) strains (5). ...
Due to genome segmentation, rotaviruses must co-package eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus NSP2 protein. While the activities of distinct RNA chaperones are well studied on smaller RNAs, little is known about their global effect on the entire viral transcriptome. Here, we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to examine the secondary structure of the rotavirus transcriptome in the presence of increasing amounts of NSP2. SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate rotavirus transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of mutation rates measured by mutational profiling reveals a global 5-fold rate increase in the presence of NSP2. We demonstrate that the normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone. Analysis of the mutation rates reveals a larger effect on stems rather than loops. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner.
... Host mRNAs have 59 caps and 39 poly(A) tails to mark them as "self," thereby preventing cytosolic degradation by host RNases. To survive these conditions, the RV guanyl and methyl transferase VP3 caps the 59 end of RV transcripts, while NSP3 binds a consensus sequence at the 39 end of each RV transcript (67)(68)(69). In addition to its role in RNA capping, VP3 N terminus also targets MAVS for degradation in a strain-specific manner ( Fig. 1), which dampens the type III IFN response in vivo (29). ...
... Competing with the host poly(A)-binding protein (PABP), NSP3 interacts with eIF4G, a component of the eIF4 ribosome recruitment complex necessary for translation (79). This protects viral RNAs from degradation, facilitating translation of viral mRNAs and inhibiting translation of host mRNAs (68,69). NSP3 has also been found to influence the host unfolded protein response, a protective feedback mechanism that restricts translation in response to an accumulation of misfolded proteins in the endoplasmic reticulum (ER) (80). ...
... Translation. NSP3 binds to a conserved consensus sequence in the RV 39 untranslated regions to facilitate translation (68,69,(146)(147)(148)(149)(150)(151). NSP3 expressed from a chimeric vaccinia virus in nonhuman primate kidney cells is sufficient to reduce host translation to levels comparable to those during RV infection (68,152,153). ...
Rotaviruses represent one of the most successful pathogens in the world, with high infectivity and efficient transmission between the young of many animal species, including humans. To overcome host defenses, rotaviruses have evolved a plethora of strategies to effectively evade the innate immune response, establish initial infection in the small intestine, produce progeny, and shed into the environment. Previously, studying the roles and relative contributions of specific rotaviral factors in innate immune evasion had been challenging without a plasmid-only reverse genetics system.
... Each dsRNA segment serves as a template for transcribing differently sized (0.67-3.4 kb) protein-coding +ssRNA transcripts (mRNAs) that can fold into regulatory structures, such as those predicted to be found in the 5′ and 3′ untranslated regions (UTRs) (4,5). In addition, consensus sequences found in 5′ and 3′ terminal regions of these segments also have important regulatory roles in ensuring proper viral infection, such as recruiting viral proteins to enhance viral translation (6,7). Li et al provided one of the most comprehensive studies of rotavirus RNAs based on the minimum free energy consensus structures deduced from multiple sequence alignments and covariation analysis of group A rotavirus (RVA) strains (5). ...
Due to genome segmentation, rotaviruses must co-package a set of eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus (RV) NSP2 protein. While the activities of distinct viral RNA chaperones are well studied on synthetic RNA substrates, little is known about their global effect on the entire viral transcriptome. Here we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to systematically examine the secondary structure of the RV transcriptome composed of eleven distinct transcripts in the absence and presence of increasing concentrations of RV NSP2. Surprisingly, SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate RV transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of the per nucleotide mutation rate measured by mutational profiling, from which SHAPE reactivities are derived, reveals a global five-fold rate increase in the presence of molar excess of NSP2. We demonstrate that the standard normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone activity. Further analysis of the mutation rate in the context of structural classification reveals a larger effect on stems rather than loop elements. Together, these data provide the first experimentally derived secondary structure model of the RV transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner, consistent with its promiscuous RNA-binding nature.
... Non-structural protein NSP3 is an RNA-binding protein associated with the conserved consensus 3 ′ -end GACC sequences of translated RV transcripts (Poncet et al., 1993(Poncet et al., , 1994. In addition, NSP3 has been recognized as an interaction partner of the translation initiation factor eIF4G1 and an eviction factor for polyA binding protein (PABP) from eIF4F. ...
Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. This review follows different stages of the viral replication cycle and summarizes highlights of structure-function studies of rotavirus-encoded proteins (both structural and non-structural), molecular mechanisms of viral replication including involvement of cellular proteins and lipids, the spectrum of viral genomic and antigenic diversity, progress in understanding of innate and acquired immune responses, and further developments of prevention of rotavirus-associated disease.
... The 7th genome segment encodes NSP3 protein [99]. The protein is slightly acidic and binds specifically the conserved tetranucleotide sequence present at the 3 0 end of the rotaviral mRNAs [100]. There is a crystal structure available for NSP3 (PDB ID:1KNZ) {Fig. ...
Rotavirus is a major cause of severe acute gastroenteritis in the infants and young children. The past decade has evidenced the role of intrinsically disordered proteins/regions (IDPs)/(IDPRs) in viral and other diseases. In general, (IDPs)/(IDPRs) are considered as dynamic conformational ensembles that devoid of a specific 3D structure, being associated with various important biological phenomena. Viruses utilize IDPs/IDPRs to survive in harsh environments, to evade the host immune system, and to highjack and manipulate host cellular proteins. The role of IDPs/IDPRs in Rotavirus biology and pathogenicity are not assessed so far, therefore, we have designed this study to deeply look at the penetrance of intrinsic disorder in rotavirus proteome consisting 12 proteins encoded by 11 segments of viral genome. Also, for all human rotaviral proteins, we have deciphered molecular recognition features (MoRFs), which are disorder based binding sites in proteins. Our study shows the wide spread of intrinsic disorder in several rotavirus proteins, primarily the nonstructural proteins NSP3, NSP4, and NSP5 that are involved in viral replication, translation, viroplasm formation and/or maturation. This study may serve as a primer for understanding the role of IDPs/MoRFs in rotavirus biology, design of alternative therapeutic strategies, and development of disorder-based drugs.
... With the exception of NSP4, all of these NSPs bind to the RNA. They have various functions in viral replication, assembly of various proteins and in viral packaging during replication [3]. ...
... The ability to monitor reaction progress has a number of advantages over end point analysis. As a result, Real-Time PCR has proven to be a powerful tool for genetic analysis [3]. DNA detection simultaneous to amplification is preferentially achieved by the use of target sequence-specific oligonucleotides linked to two different molecules, a fluorescent reporter molecule and a quenching molecule [31]. ...
Rotaviruses are classified in the genus Rotavirus and belong to family of Reoviridae, that is a family of viruses that can affect the gastrointestinal system. Reoviridae have genomes consisting of segmented, double-stranded RNA (dsRNA).This study aimed to investigate the prevalence of Rotavirus (RV) among children with acute diarrhea. One hundred and fifty pediatric patients suffering from clinical manifestation of diarrhea, fever, and vomiting were enrolled in this study. All patients underwent ELISA test for stool VP6 protein detection and real time PCR test for VP6 and NSP4 genes detection. Results showed that the frequency rate of Rotavirus infection was 33.3% by ELISA technique. The molecular techniques showed a positivity, of 33.3% for VP6 gene and 34% for NSP4 gene. The ELISA assay represented the more sensitive test in detection of Rotavirus related diarrhea in stool specimens. The results revealed that males tend to be more effected by RV than females and the bottle feeding children were more susceptible to virus infection, comparing to other feeding type. The infection rate had increased with decreasing the age of the children.
... Small interfering RNA (siRNA) experiments have shown that both the translation shutoff and nuclear export arrest are linked to the viral protein NSP3 (22,25). Rotavirus NSP3 is a viral translation enhancer and surrogate of the cellular cytoplasmic PABP (PABPC) (26) encoded by rotavirus gene 7. Upon homodimerization NSP3 recognizes the 3= end of viral mRNAs (27)(28)(29) and interacts simultaneously with the translation initiation factor eIF4G (30-32), thus strongly stimulating viral mRNA translation (24,33,34). Interaction of NSP3 with eIF4G leads to the eviction of PABPC from eIF4G (32,35) with its subsequent release from poly(A) RNA and relocalization into the nucleus (23,35,36). ...
XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an additional XBP1 RNA ( XBP1es ) that corresponded to exon-skipping in the XBP1 pre RNA was induced depending on the rotavirus strain used. We showed that the translation product of XBP1es (XBP1es) has trans-activation properties similar to those of XBP1 on ER stress response element (ERSE) containing promoters. Using mono-reassortant between ES+ ("skipping") and ES- ("nonskipping") strains of rotavirus, we showed that gene 7 encoding the viral translation enhancer NSP3 was involved in this phenomenon and that exon-skipping paralleled the nuclear relocalization of cytoplasmic PABP. We further showed, using recombinant rotaviruses carrying chimeric gene 7, that the ES+ phenotype was linked to the eIF4G-binding domain of NSP3. Because the XBP1 transcription factor is involved in stress and immunological responses, our results suggest an alternative way to activate XBP1 upon viral infection or nuclear localization of PABP.
IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. Here we show that infection with several rotavirus strains induces an alternative splicing of the RNA encoding the stressed-induced transcription factor XBP1. The genetic determinant of XBP1 splicing is the viral RNA- translation-enhancer NSP3. XBP1 being involved in cellular stress and immune responses and the XBP1 protein made from the alternatively spliced RNA being an active transcription factor, our observations raise the question of alternative splicing being a cellular response to rotavirus infection.
... [44] [32] NSP3 is bound to viral mRNAs in infected cells and it is responsible for the shutdown of cellular protein synthesis. [45] NSP3 inactivates two translation initiation factors essential for synthesis of proteins from host mRNA. First, NSP3 ejects poly(A)-binding protein (PABP) from the translation initiation factor eIF4F. ...
Rotavirus is the most common cause of diarrhoeal disease among infants and young children. It is a genus of double-stranded RNA viruses in the family Reoviridae. Nearly every child in the world is infected with rotavirus at least once by the age of five. Immunity develops with each infection, so subsequent infections are less severe; adults are rarely affected. There are eight species of this virus, referred to as A, B, C, D, E, F, G and H. Rotavirus A, the most common species, causes more than 90% of rotavirus infections in humans.
The virus is transmitted by the faecal-oral route. It infects and damages the cells that line the small intestine and causes gastroenteritis (which is often called "stomach flu" despite having no relation to influenza). Although rotavirus was discovered in 1973 by Ruth Bishop and her colleagues by electron micrograph images and accounts for approximately one third of hospitalisations for severe diarrhoea in infants and children, its importance has historically been underestimated within the public health community, particularly in developing countries. In addition to its impact on human health, rotavirus also infects animals, and is a pathogen of livestock.
Rotavirus is usually an easily managed disease of childhood, but in 2013, rotavirus caused 37 percent of deaths of children from diarrhoea and 215,000 deaths worldwide, and almost two million more become severely ill. Most of these deaths occurred in developing countries. In the United States, before initiation of the rotavirus vaccination programme, rotavirus caused about 2.7 million cases of severe gastroenteritis in children, almost 60,000 hospitalisations, and around 37 deaths each year. Following rotavirus vaccine introduction in the United States, hospitalisation rates have fallen significantly. Public health campaigns to combat rotavirus focus on providing oral rehydration therapy for infected children and vaccination to prevent the disease. The incidence and severity of rotavirus infections has declined significantly in countries that have added rotavirus vaccine to their routine childhood immunisation policies
... The occurrence of NSP3 T1 genotype on the genogroup-2 backbone in one of the study strains (RV09) is in concurrence with the results reported earlier for Japanese G9P[4] strain (S120088) (Yamamoto et al., 2015). NSP3 gene, described as interacting, extensively with host cell factors rather than with any other viral proteins, has been considered nonessential for replication in cell culture, and therefore may have a tendency to reassort more frequently as compared to other internal genes (Poncet et al., 1993;Piron et al., 1998;Heiman et al., 2008). Such reassortment involving the NSP2 N1 genotype on the genogroup-2 backbone over a period of six years has been reported for RVA strains in Japan (Doan et al., 2012). ...
Rotavirus infections associated with unusual strains are an emerging concern in rotavirus vaccination program. Recently, an increase in circulation of unusual G9P[4] strain was reported from different regions of India, placing it at the third position after G1P[8] and G2P[4], the most common rotavirus strains. The aim of the present study was to analyze complete genomic constellation of three G9P[4] strains (RV09, RV10 and RV11), determine their genetic relatedness with other genogroup-2 strains and understand the evolution of a rare E6 and other NSP4 genotypes. All strains revealed presence of genogroup-2 backbone with RV09 constituting NSP3 T1 genotype and RV10 and RV11 bearing NSP4 E6 genotype. A refined criterion adopted to classify the nine internal gene segments of G2P[4] and non-G2P[4] strains with genogroup-2 backbone into lineages and sub-lineages, indicated divergence of >8% (except NSP1: >5.5%) for lineages and >3% for sub-lineages. VP1 and/or VP3 genes of study strains showed close relationship with animal-like human rotaviruses. The estimated evolutionary rate for NSP4 E6 genotype was marginally higher (3.78×10-3 substitutions/site/year) than that of the E1 (2.6×10-3 substitutions/site/year) and E2 (3.06×10-3 substitutions/site/year) genotypes, suggesting a step towards adaptation of E6 on a genogroup-2 backbone. The time and origin of the most recent common ancestor of E6 genotype were estimated to be 1981 and South Asia respectively. Full genome and evolutionary analyses performed in this study for G9P[4] strains will help better understand the extent of gene reassortment and origin in unusual rotavirus strains that may remain viable and cause infections in humans.
... It has a molecular weight of~36 kDa, forms homodimers, and several distinct domains have been mapped to different regions of protein ( Figure 2). The N-terminal domain (aa residues 1-149) of two NSP3 monomers form an asymmetrical RNA-binding pocket that recognizes the rotaviral mRNA 3 1 GACC consensus sequence [17,[24][25][26][27], while the C-terminal domain (aa residues 206-313) binds to eIF4G in a region that overlaps with the binding site for the poly(A) binding protein (PABP) [25,26,28]. NSP3 also contains a dimerization domain in the central region of the protein (aa 150-241) that is predicted to form a coiled-coil structure allowing NSP3 dimer formation, and it has been shown that within this region there is a site of interaction with a cellular protein named RoXaN (Rotavirus X protein associated with NSP3); the function of this protein in uninfected as well as in rotavirus-infected cells is still unknown [29]. ...
The general stress and innate immune responses are closely linked and overlap at many levels. The outcomes of these responses serve to reprogram host expression patterns to prevent viral invasions. In turn, viruses counter attack these cell responses to ensure their replication. The mechanisms by which viruses attempt to control host cell responses are as varied as the number of different virus families. One of the most recurrent strategies used by viruses to control the antiviral response of the cell is to hijack the translation machinery of the host, such that viral proteins are preferentially synthesized, while the expression of the stress and antiviral responses of the cell are blocked at the translation level. Here, we will review how rotaviruses, an important agent of acute severe gastroenteritis in children, overcome the stress responses of the cell to establish a productive infectious cycle.
