The parvovirus H-1 NS2 protein affects viral gene expression through sequences in the 3' untranslated region.
We reported previously that an NS2 null mutant of parvovirus H-1 (H-1SA) was capable of lytic growth in human and hamster cells, but not in rat cells (Li and Rhode, 1991). The host-range phenotype of H-1SA was also manifested in newborn rats and was associated with a reduction of viral protein synthesis to about 10% of wild-type virus and an absence of virions in cultured rat fibroblasts. However, the H-1SA mRNAs for NS1 and capsid proteins, R1 and R3, accumulated to wild-type levels and translated well with a cell free rabbit reticulocyte lysate. These results indicate that NS2 plays an important role in the regulation of viral protein synthesis in rat cells in vivo and in vitro, but NS2 is largely dispensable in other types of cells, such as human and hamster cells. To analyze whether the 5' and 3' untranslated regions (UTR) of viral RNA are involved in the regulation by NS2, the viral VP2 gene was replaced by a reporter gene, firefly luciferase, in a plasmid clone of viral sequences and the protein synthesis under the control of P38 was evaluated by luciferase assay. Cells were transfected with luciferase expressing plasmids and subsequently infected with wild-type H-1 or H-1SA. We were able to mimic the defect in expression that we observed in cultured cells and animals with virus infection. Luciferase activity in H- 1SA-infected rat cells was about 10-fold lower than that in H-1-infected rat cells, but only 2-fold lower or less in H-1SA-infected human cells and hamster cells compared to wild-type H-1. These results are consistent with our previous data that NS2 has a host-range phenotype in the natural host of H-1, the rat. Deletion of 5' UTR sequences from P38 transcripts reduced the overall P38-luc expression but expression was NS2 independent, whereas deletion of the terminal 3' UTR sequences of viral RNA reduced NS2-dependent expression in rat cells. These results suggest that the regulation of viral protein synthesis by NS2 depends on RNA sequences in the 3' UTR.
Li
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Department of Pathology & Microbiology, University of Nebraska Medical Center, Omaha 68198.
Rhode
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D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010321:Parvoviridae / Q000235:genetics* / Q000472:pathogenicity; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D016601:RNA-Binding Proteins / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion; D013045:Species Specificity; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000097:blood / Q000235:genetics*; D014774:Virulence
D036022:Capsid Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D008156:Luciferases