Norovirus-Mediated Modification of the Translational Landscape via Virus and Host-Induced Cleavage of Translation Initiation Factors.

Noroviruses produce viral RNAs lacking a 5' cap structure and instead use a virus-encoded viral protein genome-linked (VPg) protein covalently linked to viral RNA to interact with translation initiation factors and drive viral protein synthesis. Norovirus infection results in the induction of the innate response leading to interferon stimulated gene (ISG) transcription. However, the translation of the induced ISG mRNAs is suppressed. A SILAC-based mass spectrometry approach was employed to analyze changes to protein abundance in both whole cell and m7GTP-enriched samples to demonstrate that diminished host mRNA translation correlates with changes to the composition of the eukaryotic initiation factor complex. The suppression of host ISG translation correlates with the activity of the viral protease (NS6) and the activation of cellular caspases leading to the establishment of an apoptotic environment. These results indicate that noroviruses exploit the differences between viral VPg-dependent and cellular cap-dependent translation in order to diminish the host response to infection.

The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.

UNLABELLED: All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. IMPORTANCE: Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase.

Molecular chaperone Hsp90 is a therapeutic target for noroviruses.

UNLABELLED: Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5' and 3' extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE: HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients.

Norovirus genome circularization and efficient replication are facilitated by binding of PCBP2 and hnRNP A1.

Sequences and structures within the terminal genomic regions of plus-strand RNA viruses are targets for the binding of host proteins that modulate functions such as translation, RNA replication, and encapsidation. Using murine norovirus 1 (MNV-1), we describe the presence of long-range RNA-RNA interactions that were stabilized by cellular proteins. The proteins potentially responsible for the stabilization were selected based on their ability to bind the MNV-1 genome and/or having been reported to be involved in the stabilization of RNA-RNA interactions. Cell extracts were preincubated with antibodies against the selected proteins and used for coprecipitation reactions. Extracts treated with antibodies to poly(C) binding protein 2 (PCBP2) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 significantly reduced the 5'-3' interaction. Both PCBP2 and hnRNP A1 recombinant proteins stabilized the 5'-3' interactions and formed ribonucleoprotein complexes with the 5' and 3' ends of the MNV-1 genomic RNA. Mutations within the 3' complementary sequences (CS) that disrupt the 5'-3'-end interactions resulted in a significant reduction of the viral titer, suggesting that the integrity of the 3'-end sequence and/or the lack of complementarity with the 5' end is important for efficient virus replication. Small interfering RNA-mediated knockdown of PCBP2 or hnRNP A1 resulted in a reduction in virus yield, confirming a role for the observed interactions in efficient viral replication. PCBP2 and hnRNP A1 induced the circularization of MNV-1 RNA, as revealed by electron microscopy. This study provides evidence that PCBP2 and hnRNP A1 bind to the 5' and 3' ends of the MNV-1 viral RNA and contribute to RNA circularization, playing a role in the virus life cycle.

Identification of RNA-protein interaction networks involved in the norovirus life cycle.

Human noroviruses are one of the major causes of acute gastroenteritis in the developed world, yet our understanding of their molecular mechanisms of genome translation and replication lags behind that for many RNA viruses. Due to the nonculturable nature of human noroviruses, many related members of the Caliciviridae family of small RNA viruses are often used as model systems to dissect the finer details of the norovirus life cycle. Murine norovirus (MNV) has provided one such system with which to study the basic mechanisms of norovirus translation and replication in cell culture. In this report we describe the use of riboproteomics to identify host factors that interact with the extremities of the MNV genome. This network of RNA-protein interactions contains many well-characterized host factors, including PTB, La, and DDX3, which have been shown to play a role in the life cycle of other RNA viruses. By using RNA coimmunoprecipitation, we confirmed that a number of the factors identified using riboproteomics are associated with the viral RNA during virus replication in cell culture. We further demonstrated that RNA inhibition-mediated knockdown of the intracellular levels of a number of these factors inhibits or slows norovirus replication in cell culture, allowing identification of new intracellular targets for this important group of pathogens.

Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication.

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.

Functional analysis of RNA structures present at the 3' extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence.

Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3' extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3'-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1(-/-) mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.

Functions of the 5' and 3' ends of calicivirus genomes.

The Caliciviridae family of small positive sense RNA viruses contains a diverse range of pathogens of both man and animals. The molecular mechanisms of calicivirus genome replication and translation have not been as widely studied as many other RNA viruses. With the relatively recent development of robust cell culture and reverse genetics systems for several members of the Caliciviridae family, a more in-depth analysis of the finer detail of the viral life cycle has now been obtained. As a result, the identification and characterization of the role of RNA structures in the calicivirus life cycle has also been possible. This review aims to summarize the current state of knowledge with respect to the role of RNA structures at the termini of calicivirus genomes.

Murine norovirus: propagation, quantification, and genetic manipulation.

Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. It is the most common pathogen in biomedical research colonies. MNV is also related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Like the human noroviruses, MNV is an enteric virus that replicates in the intestine and is transmitted by the fecal-oral route. MNV replicates in murine macrophages and dendritic cells in cells in culture and in the murine host. This virus is often used to study mechanisms in norovirus biology, because human noroviruses are refractory to growth in cell culture. MNV combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Herein, we describe a panel of techniques that are commonly used to study MNV biology.

Progress towards the prevention and treatment of norovirus infections.

Noroviruses are now recognized as the major cause of acute gastroenteritis in the developed world, yet our ability to prevent and control infection is limited. Recent work has highlighted that, while typically an acute infection in the population, immunocompromised patients often experience long-term infections that may last many years. This cohort of patients and those regularly exposed to infectious material, for example, care workers and others, would benefit greatly from the development of a vaccine or antiviral therapy. While a licensed vaccine or antiviral has yet to be developed, work over the past 10 years in this area has intensified and trials with a vaccine candidate have proven promising. Numerous antiviral targets and small molecule inhibitors that have efficacy in cell culture have now been identified; however, further studies in this area are required in order to make these suitable for clinical use.

Development of a strand specific real-time RT-qPCR assay for the detection and quantitation of murine norovirus RNA.

Murine norovirus (MNV), currently the only norovirus that efficiently replicates in cell culture, is often used as a model system to understand the molecular mechanisms of norovirus replication. MNV is a single stranded positive sense RNA virus of the Caliciviridae family. Replication of MNV involves the synthesis of both full length genomic and sub-genomic RNAs. The replication of these RNAs involves the synthesis of negative strand intermediates. To understand the molecular mechanism of RNA replication and the role of viral and host factors in virus replication, it is necessary to quantify accurately both positive and negative sense RNA molecules of the viral RNA during replication. Increasingly, strand specific reverse transcription-quantitative PCR (RT-qPCR) is becoming the method of choice for this kind of quantitation. Many strategies have been developed to avoid the false priming property of reverse transcriptase and to amplify specifically one strand in the presence of excess opposite strand. In this report, a SYBR based, real time RT-qPCR assay was developed to detect and quantify specifically the negative and the positive sense RNAs of MNV genomic RNA. This assay is based on using a tagged RT primer containing a non-viral sequence at the 5' end of the viral strand specific sequence. This non-viral sequence is then used to amplify selectively the strand specific cDNA at the PCR stage. This assay can be used for a range of MNV strains including MNV-1 and 3, as these are now widely accepted for use in molecular studies. The specificity of this assay was determined by its ability to quantify one strand in the presence of up to 10(6) copies of competitor opposite sense RNA. Using this assay, the production of both strands of MNV-1 RNA was monitored during viral single step growth curve.

La protein can simultaneously bind to both 3'- and 5'-noncoding regions of Japanese encephalitis virus genome.

Japanese encephalitis virus (JEV) genome is a single-stranded, positive-sense RNA with noncoding regions (NCRs) of 95 and 585 bases at its 5' and 3' ends, respectively. These may interact with viral or host proteins important for viral replication. We have previously shown that La protein binds the 3'-stem-loop (SL) structure of JEV 3'-NCR. Using electrophoretic mobility shift and ultraviolet crosslinking assays, we now show that La protein binds both 3'-SL and 5'-NCR of JEV. La protein binding to 5'-NCR RNA was stable under high salt condition (300 mM KCl) and the affinity of RNA protein interaction was high; the dissociation constant (K(d)) for La binding with 5'-NCR RNA was 8.8 nM, indicating the physiological relevance of the interaction. RNA toe-printing assays showed that La protein interacted with nucleotides located in the top loop of the predicted structure of 5'-NCR RNA. Using competitive binding studies and 5'-3' coprecipitation assay, we have demonstrated that La protein could simultaneously bind both JEV 3'- and 5'-NCRs. This may help circularize the viral genome for its efficient transcription and translation.

Polypyrimidine tract binding protein functions as a negative regulator of feline calicivirus translation.

BACKGROUND: Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5' or 3' extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5'end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle. PRINCIPAL FINDINGS: We have demonstrated that PTB interacts with at least two binding sites within the 5'end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication. CONCLUSIONS: Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.

La protein binds the predicted loop structures in the 3' non-coding region of Japanese encephalitis virus genome: role in virus replication.

Japanese encephalitis virus (JEV) genome is a single-stranded, positive-sense RNA with non-coding regions (NCRs) of 95 and 585 bases at its 5' and 3' ends, respectively. These may bind to viral or host proteins important for viral replication. It has been shown previously that three proteins of 32, 35 and 50 kDa bind the 3' stem-loop (SL) structure of the JEV 3' NCR, and one of these was identified as 36 kDa Mov34 protein. Using electrophoretic mobility-shift and UV cross-linking assays, as well as a yeast three-hybrid system, it was shown here that La protein binds to the 3' SL of JEV. The binding was stable under high-salt conditions (300 mM KCl) and the affinity of the RNA-protein interaction was high; the dissociation constant (Kd) for binding of La protein to the 3' SL was 12 nM, indicating that this RNA-protein interaction is physiologically plausible. Only the N-terminal half of La protein containing RNA recognition motifs 1 and 2 interacted with JEV RNA. An RNA toe-printing assay followed by deletion mutagenesis showed that La protein bound to predicted loop structures in the 3' SL RNA. Furthermore, it was shown that small interfering RNA-mediated downregulation of La protein resulted in repression of JEV replication in cultured cells.