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. The genomes of small positive-strand RNA viruses must not only encode the viral proteins required for viral genome replication and infectious virus production, but they must also contain cis-acting RNA sequences and structures that play critical roles in the virus life cycle (40). Host cell factors that interact with these RNA elements play important roles in all aspects of the virus life cycle (49); consequently, the manipulation of these interactions via the introduction of mutations into the viral genome is a viable approach to rational attenuation (24,26,57,78). In addition, the modification of these RNA-protein interactions by using small-molecule or peptide inhibitors has been shown to provide antiviral activity, at least in cell culture (8,19).Noroviruses, first identified in 1972 by Albert Kapikian (33), are now accepted as the major cause of viral gastroenteritis in the developed world (21). Reports indicate that in excess of 23 million cases occur each year in the United States, resulting in a significant economic impact. Now over 20 years since the first full genome sequence of a norovirus became available (77), investigators are still unable to efficiently propagate human noroviruses in cell culture (15, 37). Recent data have indicated that the viral RNA purified from the feces of individuals infected with Norwalk virus, the prototype human norovirus, can replicate when transfected into immortalized cells in culture (23). These data clearly demonstrate that one of the major blocks in the ability of human noroviruses to replicate in cell culture is the ability of the virus to enter and spread from cell to cell. It is also clear that the intracellular environ...

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. Curr.

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 (RTqPCR) 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 RTqPCR 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 nonviral 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.

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.