Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Abstract

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA͞ T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.human norovirus ͉ norovirus RNA replication N oncultivatable human norovirus belongs to the Norovirus genus of the family Caliciviridae and is classified as a Group B biodefense pathogen. Human noroviruses are responsible for almost all outbreaks (Ͼ95%) of nonbacterial gastroenteritis in the United States and Europe (1, 2). Incidents of the rapid spread of norovirus-related illness in the cruise ship industry have led to an increased recognition of the impact of norovirus infections on public health (3).Norwalk virus (NV), the prototype strain of human norovirus, is a nonenveloped virus that contains a Ϸ7.7-kb positive-sense single-stranded RNA genome (4). The NV genome is polyadenylated at its 3Ј end and encodes three primary ORFs (Fig. 1A) (4). ORF1 encodes a nonstructural polyprotein containing the following genes from the 5Ј end to the 3Ј end, respectively: p48, nucleotide triphosphatase (NTPase), p22, VPg, protease (Pro), and RNA polymerase (RNA Pol) (Fig. 1 A) (5). ORF2 and ORF3 encode structural proteins, the major capsid protein (VP1) and a minor structural protein (VP2), respectively ( Fig. 1 A) (4). Expression of VP1 in insect cells infected with baculovirus recombinants results in the self-assembly of empty recombinant virus-like particles (VLPs) (6, 7). VP2 is also incorporated into VLPs when VP2 is coexpressed with VP1 (8), and its presence increases the yield and stability of VLPs (9).Since the initial cloning and sequencing of the NV genome (4, 10), many other norovirus sequences have been reported (11,12). Despite ...

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

“…First, they show that authentic U201 RNA is produced and that the sequence of the subgenomic RNA generated by this reverse genetics system is identical to the predicted native subgenomic RNA. This finding confirms results from the MVA/T7 system that expressed the NV genome (10). Second, they show that progeny virus with a similar density to native virions (11) contains not only the predicted VP1, VP2, and genomic RNA but also, VPg (Fig.…”

“…Gnotobiotic pigs can support replication of a HuNoV genogroup II (GII) strain with the occurrence of mild diarrhea, fecal virus shedding, and immunofluorescent (IF) detection of both structural and nonstructural proteins in enterocytes (9). Previous systems to express the HuNoV genome from cloned DNA using T7/vaccinia systems showed that mammalian cells can produce progeny virus (10,11), but these systems are not sufficiently efficient to be widely used to propagate HuNoVs in vitro. The factors responsible for the block(s) of viral replication using standard cell culture systems remain unknown.…”

“…A 3′ coterminal polyadenylated subgenomic RNA is produced within infected cells. Both genomic and subgenomic RNAs have the same nucleotide sequence motif at their 5′ ends, and they are believed for HuNoVs and shown for MNV to be covalently linked to the nonstructural protein VPg at the 5′ ends (10,13). During MNV infection of cells, nonstructural proteins are expressed from genomic RNA and form an RNA replication complex that generates new genomic RNA molecules as well as subgenomic RNAs encoding VP1, VP2, and the unique protein called VF1 (14).…”

Porcine enteric caliciviruses: Genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology

“…The pathogenesis of porcine SaV Cowden strain was delineated in gnotobiotic pigs [19,20]. Most recently, reverse genetic systems were generated for the porcine Cowden SaV and the human Norwalk NoV, in which infectious viral particles were rescued after replication of the viral genomic RNA [35,36]. In spite of these advances, most NoVs and SaVs remain refractory to growth in cell culture and extensive efforts to propagate them in vitro have failed [37].…”

Caliciviruses Differ in Their Functional Requirements for eIF4F Components

“…The human caliciviruses, including the prototype Norwalk virus, have yet to be fully propagated in tissue culture, although recent results suggest that limited genome replication and encapsidation can occur using a vaccinia virus-driven expression system (12). In contrast to the human caliciviruses, feline calicivirus (FCV), porcine enteric calicivirus (13,14), and murine norovirus 1 (MNV) (15) can be propagated in tissue culture.…”