2nd National Conference Human Retroviruses and Related Infections Washington, DC - January 29 - February 2, 1995

Natl Conf Hum Retrovir Relat Infect 1995 Jan 29-Feb 2;2: (abstract no. 28)

Lang SM, Jung JU, Desrosiers RC
New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772-9102

The nef gene of the pathogenic molecular clone SIVmac239/nef-open encodes a 34 kDa phosphoprotein. Although not necessary for virus replication in a variety of standard cell culture systems, the nef gene of SIVmac239 is dramatically important for viral replication in rhesus monkeys. To study the molecular basis of nef action, we stably expressed SIVmac239 nef protein in the CEM×174 cell line using the retroviral vector pLXSN and an amphotropic packaging plasmid. As has bee reported previously by others, we observed downregulation of surface cd4 specifically in cells expressing full-length nef protein. Changes in CD4 surface expression were not observed in control cell lines carrying the nef gene in antisense orientation or in cells carrying a truncated nef. Using a SIV nef-specific polyclonal rabbit antiserum, nef immunocomplexes were precipitated from the nef-expressing CEM×174 cells. Assay of in vitro kinase activity revealed the specific phosphorylation of 5 proteins migrating with mobilities of 34, 65, 80, 110 and 130 kDa by SDS-PAGE. The 34 kDa protein that was the target of phosphorylation products were observed in COS cells using transient expression of nef from SIVmac239. These results, and the recent report of Sawai et al. (Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1539-43), suggest that nef may activate cellular protein kinases. Progress on identification of the cellular protein kinase will be presented.

Keywords: Animals, Antigens, CD4, COS Cells, Cell Line, Down-Regulation, Gene Products, nef, Genes, nef, In Vitro, Macaca mulatta, Phosphorylation, Protein Kinases, Protein-Serine-Threonine Kinases, Proteins, Rabbits, Simian immunodeficiency virus, Virus Replication, enzymology, genetics, immunology, virology

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1995-01-29
28

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