Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Abstract

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1,2 . During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3 . The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6,7 .MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) ( Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8 .Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery [9][10][11] . Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs [12][13][14] . This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches.Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis [15][16][17] . Video LinkThe video component of this article can be found at https://www.jove.com/video/4145/ Protocol RNA Transcription and Capping for The Recovery of Infectious MNVThis protocol is designed to allow the efficient recovery of infectious MNV from cDNA via in vitro transcription and its subsequent in vitro capping (section 1.1). The resulting capped transcripts are then transfected into cells to recover infectious MNV (sections 1.2 and ...

Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA

“…In vitro-transcribed RNA-based systems have recently been successful for several animal NoVs and caliciviruses, with variation in the requirement for capped or uncapped RNA [FCV (29,30), PEC (26), MNV (15,31), RHDV (32), and Tulane virus (33)]. Here, we report a generalizable reverse genetics system for HuNoVs that uses the EF-1α promoter and viral cDNA to produce progeny virus containing infectious RNA as well as has the ability to recover virus containing a reporter GFP gene.…”

“…Previous reverse genetics systems for HuNoV used helper vaccinia MVA/T7 virus-based systems. Although helper virus-free systems have been developed for MNV (15,16), no such system is available for HuNoVs. To overcome these problems, we established a reverse genetics system driven by a mammalian elongation factor-1α…”

Norovirus Translation Requires an Interaction between the C Terminus of the Genome-linked Viral Protein VPg and Eukaryotic Translation Initiation Factor 4G

“…Reverse genetics recovery of recombinant MNV was performed by infecting baby hamster kidney cells with fowlpox virus expressing T7 RNA polymerase followed by transfection of MNV cDNA expression constructs as described previously (22, 34). To analyze protein expression, cells were harvested 24 h post-transfection for Western blot analysis.…”

The Murine Norovirus Core Subgenomic RNA Promoter Consists of a Stable Stem-Loop That Can Direct Accurate Initiation of RNA Synthesis

“…Despite their impact, noroviruses remain poorly characterized, as an in-depth understanding of the molecular mechanisms of norovirus genome translation and replication has been hampered by the inability to culture human norovirus (3). Murine norovirus (MNV) is a model system with which to understand the norovirus life cycle (4), as it replicates in cultured cells (5) and a number of tractable reverse genetics systems are available (6)(7)(8)(9). Studies of MNV have therefore led to a number of significant advances in the understanding of the molecular mechanism of norovirus genome translation and replication (reviewed in reference 10).…”