Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells

Abstract

Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase ( Noroviruses, members of the family Caliciviridae, are the major etiologic agents of nonbacterial epidemic gastroenteritis (13,27,30,46,48). The lack of a cell culture system for human pathogens has necessitated a recombinant DNA-based approach for the classification of circulating strains, the generation of diagnostic tests, and the elucidation of a proposed virus replication strategy. The human noroviruses segregate into three major genogroups (designated GI, GII, and GIV), with multiple genetic clusters, or genotypes, defined within each genogroup (2, 18, 52). The 7.6-to 7.7-kb RNA genome of the noroviruses is organized into three separate open reading frames (ORFs) (ORFs 1, 2, and 3) (17, 23). ORF1 encodes a large nonstructural polyprotein that is processed by the virusencoded 3C-like (3CL) proteinase (Pro) into the mature nonstructural proteins (17, 23, 25). ORF2 encodes the major capsid protein, VP1, and ORF3 encodes a minor structural protein, VP2 (11,17,23).In in vitro experiments, the human norovirus 3CL Pro recognized five cleavage sites within ORF1 with various efficiencies to release six mature cleavage products with the gene order Nterm-NTPase-p20/p22-VPg-Pro-polymerase (Pol) (4,5,15,25,26,39). In addition, several intermediate precursor products were observed in in vitro studies, but their presence during norovirus replication could not be confirmed in the absence of a cell culture system (4, 25).The recent identification of murine norovirus (MNV) and the discovery that it can be propagated in a murine macrophage-like cell line (RAW264.7 [RAW]) provided the first cell culture system to study the molecular mechanisms of norovirus replication (20, 51). The MNV (murine norovirus 1 [MNV-1]) RNA genome is 7,382 nucleotides (nt) in length, with the coding region flanked by a short, 5-nt sequence at the 5Ј end and a 75-nt sequence at the 3Ј end followed by a poly(A) tail (20). The MNV genome is organized into three ORFs with a predicted gene order identical to that of human noroviruses. However, MNV is sufficiently divergent in its sequence to warrant its segregation into a new norovirus genogroup, designated GV (20,52). Northern blot analysis of MNV-infected cells showed the presence of two major positive-strand RNA species corresponding to the genomic and subgenomic RNA, which is characteristic of calicivirus positive-strand RNA replication (51).The goal of our present work was to define the cleavage map of the MNV nonstructural polyprotein and examine proteolytic processing of norovirus proteins during an authentic infection in cell culture. We demonstrate that the MNV 3CL Pro can mediate the cleavage of the MNV nonstructural po...

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

“…This result illustrates one level of possible regulation of expression in this system. Demonstration that the mature HuNoV protease can function in trans in cells confirms results with other NoV and calicivirus systems that tested trans protease activity in bacterial or in vitro cell-free translation systems (18)(19)(20)(35)(36)(37)(38) or cells transfected with ORF1 constructs (39). They also suggest that establishment of a cell line that expresses a mutant full-length HuNoV ORF1 construct that includes a reporter protein that would be released for detection after cleavage could lead to a useful assay to detect superinfection with an infectious HuNoV with active protease that could release the reporter.…”

“…1A indicates the ORF1 proteins using their names based on function and nonstructural protein numeric designations) (18)(19)(20). To assess ORF1 synthesis and polyprotein cleavage, Western blot analysis was performed at 24 h posttransfection (hpt) (Fig.…”

Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

“…The mature proteins include an RNA-dependent RNA polymerase (RdRp) (NS7 pol ), an ATPase (NS3), and proteins that disrupt cellular trafficking (NS1-2 and NS4) (18)(19)(20)(21)(22)(23).…”

Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway

“…The genome is organized into four open reading frames (ORF-1-4). ORF-1 encodes the non-structural proteins, whereas ORF-2 and ORF-3 encode structural proteins (Sosnovtsev et al, 2006). ORF-4 was recently identified within the MNV-1 genome and encodes a single protein of unknown function (Thackray et al, 2007).…”