Structure and function of rotavirus nonstructural protein NSP3

Poncet, Didier; Aponte, Carlos; Cohen, Jean

doi:10.1007/978-3-7091-6553-9_4

Cited by 10 publications

(6 citation statements)

References 16 publications

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“…In the present study, the primers and probe of the real time RT-PCR were selected from a strictly-conserved region, NSP3, which plays an important role in rotavirus replication [Poncet et al, 1996]. Homologous sequences for these primers and probes were found in bovine, simian, porcine, and human group A rotaviruses.…”

Section: Discussionmentioning

confidence: 99%

Increased detection of rotavirus using a real time reverse transcription‐polymerase chain reaction (RT‐PCR) assay in stool specimens from children with diarrhea

Pang

Lee

Boroumand

et al. 2004

Journal of Medical Virology

200

110

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Six-hundred and twenty-six stool specimens collected from children with diarrhea over a 12-month period were tested for rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay, a conventional nested PCR assay and by electron microscopy (EM). A fragment of 87 bp in a highly-conserved region of non-structural protein 3 (NSP3) in rotavirus genome was amplified by a single-step RT-PCR protocol in a closed-tube system. Rotavirus was detected in 123 samples (20%) with the real time RT-PCR assay, 113 samples (18%) with the nested-PCR assay, and 79 samples (13%) with EM. Using serial diluted nucleic acid extract, we compared the sensitivity of real time RT-PCR with conventional RT-PCR and conventional nested PCR assays. Real time RT-PCR was two to four logs more sensitive than the conventional assays. The reaction time required for the RT-PCR assay is about half the time required for the conventional nested-PCR. The real time RT-PCR assay is both simple and rapid with advantages including enhanced sensitivity and a lower risk for cross-contamination making it a useful tool for the detection of rotavirus in various situations including sporadic gastroenteritis, outbreaks, and environmental investigations. G(1) was the predominant type (89%), followed by G(2) (10%), and G(4) (1%). No rotavirus of G(3), G(8), and G(9) types were found. The peak season for rotavirus infection was January to May in northern Alberta.

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Section: Discussionmentioning

confidence: 99%

Increased detection of rotavirus using a real time reverse transcription‐polymerase chain reaction (RT‐PCR) assay in stool specimens from children with diarrhea

Pang

Lee

Boroumand

et al. 2004

Journal of Medical Virology

200

110

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“…Other small, phosphorylated viral proteins have been reported to enhance translation. For example, NSP3, a nonstructural phosphorylated protein from rotaviruses, has been shown to enhance translation of viral RNAs by binding as a dimer to the 3Ј end of the viral RNAs and likely circularizing the RNA (46)(47)(48). It has also been proposed that the 35K form of VP2 is involved in encapsidation of viral RNA.…”

Section: Discussionmentioning

confidence: 99%

The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein

Bertolotti-Ciarlet

Crawford

Hutson

et al. 2003

J Virol

186

133

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Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3 untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3 terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3 elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3UTR (ORF2؉3؉3UTR). In contrast, expression of VP1 from constructs that lacked the 3UTR (ORF2؉3), ORF3 (ORF2؉3UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3UTR revealed that the 3UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.The Norwalk virus (NV) is a member of the genus Norovirus in the family Caliciviridae. The recent accessibility of molecular diagnostic assays has demonstrated that noroviruses are the leading cause of outbreaks of nonbacterial gastroenteritis worldwide (17). In addition, caliciviruses have been recently classified as category B biodefense pathogens (http://www.niaid .nih.gov/biodefense/). The NV 7.7-kb positive-strand RNA genome is organized into a 5Ј untranslated region (UTR), three open reading frames (ORFs), a 3ЈUTR, and a poly(A) tail (19). The NV predicted subgenomic RNA contains ORF2, ORF3, and the 3ЈUTR. ORF2 and ORF3 code for the major capsid protein (VP1) and a small basic structural protein (VP2), respectively. The ORF3 protein (VP2) has been detected in NV virions and virus-like particles (VLPs) (15). VP2 or a VP2-equivalent protein has been detected in the virions of other members of the Caliciviridae family such as the rabbit hemorrhagic disease virus (RHDV) (34) and feline calicivirus (FCV) (54). VP2 is not essential for the formation of NV VLPs because baculovirus recombinants expressing VP1 alone are able to produce NV VLPs (5, 58). The highly basic VP2 may serve a function similar to the basic N terminus of plant virus capsid proteins, which are involved in RNA binding, since the N terminus of NV VP1 is acidic and is not thought to ...

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“…Also, the RNA-dependent RNA polymerase of influenza A virus exhibits structural specificity for an internal RNA loop of the viral promoter (42). However, RNA-binding proteins of none of the other members of Reoviridae family, such as reovirus or rotavirus have been shown to have RNA structural specificity, although rotavirus NSP3 appears to bind a linear sequence found at the 3Ј end of all rotavirus RNA segments (43).…”

Section: Discussionmentioning

confidence: 99%

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

Lymperopoulos

Wirblich

Brierley

et al. 2003

Journal of Biological Chemistry

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The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.Viruses that have a segmented RNA genome face a challenging task in the recruitment and assortment of specific viralcoded RNA species in the infected cells. It is commonly believed that each of the viral segments must possess some specific sequence or structures, which is recognized by one or more virally encoded proteins to facilitate these processes. Generally, viral RNA-binding proteins are distinguished by being in one of two categories. The first includes proteins that are associated with the nucleocapsid (nucleoproteins) (1) and are involved in the replication process (transcription and packaging) of the viral genome (2, 3). The second includes proteins that play an essential role in recruiting, transporting (4), modifying (5), and translating (6) viral RNA. These proteins can also interact with cellular RNA to suppress the expression of regulatory genes (7), so protecting the viral RNA from cellular recognition mechanisms to use the cellular machinery for virus propagation (6). Members of the Reoviridae have segmented double-stranded RNA genomes enclosed within the double capsids of the virions. During virus entry into the host cells the outer capsid proteins are lost, allowing the viral core to initiate the transcription of genomic RNAs. The newly synthesized single-stranded RNA species are subsequently released into the cytosol and in turn serve both as templates for viral doublestranded RNA genome synthesis and also act as messengers for the synthesis of viral proteins within the cytoplasm (see review, see Ref. 8). However, to date it is not known how the 10 -12 RNA segments are specifically recruited and transported to the virus replication and assembly sites within the cytoplasm and if any specific sequence is involved. Bluetongue virus is an o...

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Structure and function of rotavirus nonstructural protein NSP3

Cited by 10 publications

References 16 publications

Increased detection of rotavirus using a real time reverse transcription‐polymerase chain reaction (RT‐PCR) assay in stool specimens from children with diarrhea

Increased detection of rotavirus using a real time reverse transcription‐polymerase chain reaction (RT‐PCR) assay in stool specimens from children with diarrhea

The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

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