Variation in ORF3 of genogroup 2 Norwalk-like viruses

Seah, Ee Ling; Gunesekere, Ishara C; Marshall, John A.; Wright, P.J.

doi:10.1007/s007050050563

Cited by 17 publications

(16 citation statements)

References 14 publications

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“…Overall, both the separate and combined alignments of the SLV and NLV sequences showed relatively good concordance with those previously reported [3,13,16,18,27,35,42]. However, the alignment of 10 SLV sequences indicated marked differences in the amino acid and nucleotide sequences of the 3 end of the 3 ORF and the 5 end of the 3 UTR of London/92 strain compared to the remaining 9 SLV strains.…”

Section: Alignment Of Sequencessupporting

confidence: 86%

“…Bootstrap values are indicated as % of 100 replicates. Genogroups and genetic clusters were determined in the present study, based on the analysis of the entire capsid sequences; those of NLVs refer to results of the previous [1,35] and the current studies Table 3. Pairwise distances among capsid overlap ORF sequences from genogroup I SLV strains.…”

Section: Utrmentioning

confidence: 99%

“…Other SLV and NLV sequences were available in GenBank. Genogroups and genetic clusters of SLVs were determined in this study; those of NLVs refer to results of previous [35,1] Amplification and sequencing of a 3-kb region at the 3 end of the genome…”

Section: Introductionmentioning

confidence: 99%

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Genetic classification of "Sapporo-like viruses"

Schuffenecker

Ando

Thouvenot

et al. 2001

Archives of Virology

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Section: Alignment Of Sequencessupporting

confidence: 86%

Section: Utrmentioning

confidence: 99%

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Genetic classification of "Sapporo-like viruses"

Schuffenecker

Ando

Thouvenot

et al. 2001

Archives of Virology

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“…The synthesis of the ORF 3 protein in a cell-free translation system has been reported for FCV and Camberwell virus (24,54). Considerable sequence variability of ORF 3 among members of the Norwalk-like viruses (NLVs) has been re-ported and is consistent with the idea that ORF 3 might be a structural protein (42,45).…”

supporting

confidence: 53%

Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein

Glass

White

Ball

et al. 2000

J Virol

249

190

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Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3 end of the genome beginning with the ORF 2 coding sequences (ORFs 2؉3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3 end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple highermolecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

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“…1 A) that is cleaved into the corresponding nonstructural proteins by Pro (4,(20)(21)(22)(23)(24)(25), detection of the correct sizes of Pro and RNA Pol, located at the extreme 3Ј end of ORF1, by radioimmunoprecipitation indicates that the nonstructural polyprotein was translated from the genomic RNA expressed in 293T cells, and the individual nonstructural proteins were subsequently generated by cleavage with a biologically active Pro. Both Pro and RNA Pol also were detected by immunofluorescence in the cytoplasm of the expressing cells.…”

Section: Strategy For Expression Of the Native Forms Of Nv Genomic Andmentioning

confidence: 99%

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Asanaka

Atmar

Ruvolo

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

102

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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 ...

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Variation in ORF3 of genogroup 2 Norwalk-like viruses

Cited by 17 publications

References 14 publications

Genetic classification of "Sapporo-like viruses"

Genetic classification of "Sapporo-like viruses"

Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

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