Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity.
A consensus sequence in parvovirus nonstructural protein NS1 has been predicted to be an ATP-binding domain associated with an ATPase and a DNA helicase activity. To investigate the function of NS1 in viral gene expression, a site-directed mutagenesis converting NS1 lysine 405 to serine in parvovirus H-1 was carried out by the polymerase chain reaction. As shown previously, a parvovirus genome containing a deleted NS1 gene was excised from a bacterial plasmid and replicated when a wild-type NS1 gene was provided in trans but failed to be excised and replicate when the mutant NS1 gene was supplied. Interestingly, the serine 405 mutation totally lost the activity of trans activation on the virus late promoter (P38) in a chloramphenicol acetyltransferase (CAT) assay and it lost evidence for cytotoxicity in two tumor cell lines (HeLa Gey and NB324K). The serine 405 NS1 protein was translocated normally to the nucleus. These results suggest that the NS1 lysine 405 of H-1 in its putative purine nucleotide-binding site is essential for viral DNA replication and that this domain may be involved in the regulation of the P38 promoter by an unknown mechanism. The loss of NS1 cytotoxicity on tumor cells suggests that NS1 expression is the major cause of cell killing by parvoviruses, which may facilitate further study of the mechanism of oncosuppression by parvoviruses.
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Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198.
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D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002472:Cell Transformation, Viral; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D006801:Humans; D008239:Lysine; D009154:Mutation; D010321:Parvoviridae / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012694:Serine; D015533:Transcriptional Activation; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics* / Q000378:metabolism; D017361:Viral Nonstructural Proteins
D004279:DNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D012694:Serine; D008239:Lysine