Norovirus RNA Synthesis is Modulated by an Interaction Between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1
ABSTRACT Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity, but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (Subba-Reddy et al., 2011. J. Virol., 85:13027-13037). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism in virus replication. We determined that the GII.4 and MNV VP1 proteins could enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from the GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs could bind to the purified RdRps in vitro. A MNV replicon with a frameshift mutation in the ORF2 that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time RT-qPCR. Trans-complementation of VP1 or its SD significantly recovered the VP1-knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.
SourceAvailable from: Morgan Reece Herod[Show abstract] [Hide abstract]
ABSTRACT: Investigations into human norovirus infection, replication and pathogenesis, as well as the development of potential antiviral agents, have been restricted by the lack of a cell culture system for human norovirus. To date, the optimal cell culture surrogate virus model for studying human norovirus biology is the murine norovirus (MNV). In this report we generate a tetracycline-regulated, inducible eukaryotic cell system expressing the entire MNV ORF1 polyprotein. Once induced, the MNV ORF1 polyprotein was faithfully processed to the six mature non-structural proteins that predominately located to a discrete perinuclear region, as has been observed in active MNV infection. Furthermore, we found that expression of the ORF1 polyprotein alone was sufficient to induce apoptosis, characterised by caspase-9 activation and survivin down-regulation. This cell line provides a valuable new tool for studying MNV ORF1 non-structural protein function, screening for potential antiviral agents and acts as a proof-of-principle for such systems to be developed for human noroviruses.PLoS ONE 03/2014; 9(3):e90679. DOI:10.1371/journal.pone.0090679 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Viral gastroenteritis is one of the most common diseases in humans, and it is primarily caused by rotaviruses (RVs), astroviruses (AstVs), adenoviruses (AdVs), noroviruses (NoVs), and sapoviruses (SaVs). In this study, we determined the distribution of viral gastroenteritis and human calicivirus (HuCVs) in acute gastroenteritis patients in Shenzhen, China, during 2011. Real-time RT-PCR was used to detect norovirus (NoV), group A rotavirus (RV), adenovirus (AdV), and astrovirus (AstV). From a total of 983 fecal samples, NoV was detected in 210 (21.4 %); RoV in 173 (17.6 %); AstV in 10 (1.0 %); and AdV in 15 (1.5 %). Mixed infections involving two NoVs were found in 21 of the 387 pathogen-positive stool specimens. NoV and SaV genotypes were further tested using RT-PCRs and molecular typing and phylogenetic analysis were then performed based on the ORF1-ORF2 region for NoV and a conserved nucleotide sequence in the capsid gene for SaV. Of the 68 typed strains that were sequenced and genotyped, five were NoV G1 (7.5 %) and 63 were NoV GII (96.6 %). GII strains were clustered into five genotypes, including GII.4 (65.1 %; 36 GII.4 2006b and five GII.4 New Orleans), GII.3 (28.6 %), GII.2 (3.2 %), GII.6 (1.6 %), and GII.1 (1.6 %). While all fecal specimens were tested for SaVs, 15 (1.5 %) were positive, and of these, 12 isolates belonged to G1.2, and the remaining three SaV strains belonged to the SaV GII genogroup. Although various HuCVs were detected in acute gastroenteritis patients, NoV GII.4 2006b was more prevalent than the other HuCVs.Archives of Virology 03/2014; 159(8). DOI:10.1007/s00705-014-1986-6 · 2.28 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Caliciviruses use reinitiation of translation governed by a 'termination upstream ribosomal binding site' (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.PLoS ONE 07/2014; 9(7):e102254. DOI:10.1371/journal.pone.0102254 · 3.53 Impact Factor