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. 1990 Oct;64(10):4654–4660. doi: 10.1128/jvi.64.10.4654-4660.1990

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.

X Li 1, S L Rhode 3rd 1
PMCID: PMC247949  PMID: 2144594

Abstract

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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anton I. A., Lane D. P. Non-structural protein 1 of parvoviruses: homology to purine nucleotide using proteins and early proteins of papovaviruses. Nucleic Acids Res. 1986 Oct 10;14(19):7813–7813. doi: 10.1093/nar/14.19.7813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berns K. I., Kotin R. M., Labow M. A. Regulation of adeno-associated virus DNA replication. Biochim Biophys Acta. 1988 Dec 20;951(2-3):425–429. doi: 10.1016/0167-4781(88)90116-9. [DOI] [PubMed] [Google Scholar]
  3. Bradley M. K., Smith T. F., Lathrop R. H., Livingston D. M., Webster T. A. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4026–4030. doi: 10.1073/pnas.84.12.4026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brandenburger A., Legendre D., Avalosse B., Rommelaere J. NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp. Virology. 1990 Feb;174(2):576–584. doi: 10.1016/0042-6822(90)90110-d. [DOI] [PubMed] [Google Scholar]
  5. Campione-Piccardo J. Software library with applications in virology and molecular biology. J Virol Methods. 1986 Jun;13(3):191–195. doi: 10.1016/0166-0934(86)90013-3. [DOI] [PubMed] [Google Scholar]
  6. Chejanovsky N., Carter B. J. Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication. J Virol. 1990 Apr;64(4):1764–1770. doi: 10.1128/jvi.64.4.1764-1770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clertant P., Cuzin F. Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses. J Biol Chem. 1982 Jun 10;257(11):6300–6305. [PubMed] [Google Scholar]
  8. Cotmore S. F., Tattersall P. The NS-1 polypeptide of minute virus of mice is covalently attached to the 5' termini of duplex replicative-form DNA and progeny single strands. J Virol. 1988 Mar;62(3):851–860. doi: 10.1128/jvi.62.3.851-860.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cotmore S. F., Tattersall P. The autonomously replicating parvoviruses of vertebrates. Adv Virus Res. 1987;33:91–174. doi: 10.1016/s0065-3527(08)60317-6. [DOI] [PubMed] [Google Scholar]
  10. Geider K., Hoffmann-Berling H. Proteins controlling the helical structure of DNA. Annu Rev Biochem. 1981;50:233–260. doi: 10.1146/annurev.bi.50.070181.001313. [DOI] [PubMed] [Google Scholar]
  11. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hirling H., Scheffner M., Restle T., Stahl H. RNA helicase activity associated with the human p68 protein. Nature. 1989 Jun 15;339(6225):562–564. doi: 10.1038/339562a0. [DOI] [PubMed] [Google Scholar]
  13. Im D. S., Muzyczka N. Factors that bind to adeno-associated virus terminal repeats. J Virol. 1989 Jul;63(7):3095–3104. doi: 10.1128/jvi.63.7.3095-3104.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Im D. S., Muzyczka N. The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell. 1990 May 4;61(3):447–457. doi: 10.1016/0092-8674(90)90526-k. [DOI] [PubMed] [Google Scholar]
  15. Labieniec-Pintel L., Pintel D. The minute virus of mice P39 transcription unit can encode both capsid proteins. J Virol. 1986 Mar;57(3):1163–1167. doi: 10.1128/jvi.57.3.1163-1167.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Labow M. A., Graf L. H., Jr, Berns K. I. Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes. Mol Cell Biol. 1987 Apr;7(4):1320–1325. doi: 10.1128/mcb.7.4.1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Labow M. A., Hermonat P. L., Berns K. I. Positive and negative autoregulation of the adeno-associated virus type 2 genome. J Virol. 1986 Oct;60(1):251–258. doi: 10.1128/jvi.60.1.251-258.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pintel D., Merchlinsky M. J., Ward D. C. Expression of minute virus of mice structural proteins in murine cell lines transformed by bovine papillomavirus-minute virus of mice plasmid chimera. J Virol. 1984 Nov;52(2):320–327. doi: 10.1128/jvi.52.2.320-327.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rhode S. L., 3rd Both excision and replication of cloned autonomous parvovirus DNA require the NS1 (rep) protein. J Virol. 1989 Oct;63(10):4249–4256. doi: 10.1128/jvi.63.10.4249-4256.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rhode S. L., 3rd Construction of a genetic switch for inducible trans-activation of gene expression in eucaryotic cells. J Virol. 1987 May;61(5):1448–1456. doi: 10.1128/jvi.61.5.1448-1456.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rhode S. L., 3rd Replication process of the parvovirus H-1 V. Isolation and characterization of temperature-sensitive H-1 mutants. J Virol. 1976 Feb;17(2):659–667. doi: 10.1128/jvi.17.2.659-667.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rhode S. L., 3rd, Richard S. M. Characterization of the trans-activation-responsive element of the parvovirus H-1 P38 promoter. J Virol. 1987 Sep;61(9):2807–2815. doi: 10.1128/jvi.61.9.2807-2815.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rhode S. L., 3rd trans-Activation of parvovirus P38 promoter by the 76K noncapsid protein. J Virol. 1985 Sep;55(3):886–889. doi: 10.1128/jvi.55.3.886-889.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. SHEIN H. M., ENDERS J. F. Multiplication and cytopathogenicity of Simian vacuolating virus 40 in cultures of human tissues. Proc Soc Exp Biol Med. 1962 Mar;109:495–500. doi: 10.3181/00379727-109-27246. [DOI] [PubMed] [Google Scholar]
  25. Scheffner M., Knippers R., Stahl H. RNA unwinding activity of SV40 large T antigen. Cell. 1989 Jun 16;57(6):955–963. doi: 10.1016/0092-8674(89)90334-6. [DOI] [PubMed] [Google Scholar]
  26. Snyder R. O., Samulski R. J., Muzyczka N. In vitro resolution of covalently joined AAV chromosome ends. Cell. 1990 Jan 12;60(1):105–113. doi: 10.1016/0092-8674(90)90720-y. [DOI] [PubMed] [Google Scholar]
  27. Sopta M., Burton Z. F., Greenblatt J. Structure and associated DNA-helicase activity of a general transcription initiation factor that binds to RNA polymerase II. Nature. 1989 Oct 5;341(6241):410–414. doi: 10.1038/341410a0. [DOI] [PubMed] [Google Scholar]
  28. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  29. Sung P., Higgins D., Prakash L., Prakash S. Mutation of lysine-48 to arginine in the yeast RAD3 protein abolishes its ATPase and DNA helicase activities but not the ability to bind ATP. EMBO J. 1988 Oct;7(10):3263–3269. doi: 10.1002/j.1460-2075.1988.tb03193.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tratschin J. D., Miller I. L., Carter B. J. Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol. 1984 Sep;51(3):611–619. doi: 10.1128/jvi.51.3.611-619.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tratschin J. D., Tal J., Carter B. J. Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product. Mol Cell Biol. 1986 Aug;6(8):2884–2894. doi: 10.1128/mcb.6.8.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. de la Maza L. M., Carter B. J. Inhibition of adenovirus oncogenicity in hamsters by adeno-associated virus DNA. J Natl Cancer Inst. 1981 Dec;67(6):1323–1326. [PubMed] [Google Scholar]

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