Abstract
Among the primate lentiviruses (human immunodeficiency virus (HIV) -1, HIV-2, and simian immunodeficiency virus (SIV), the nef gene is highly conserved and encodes a myristylated protein of approximately 27 kDa (HIV-1) or approximately 34 kDa (HIV-2, SIV). Previously, we found Nef expressed either as a CD8-Nef fusion protein or as a native protein in virally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. Using transient expression, various Nef alleles and mutants have been analyzed for association with the cellular kinase activity. The ability of Nef to associate with the kinase activity is conserved among several alleles of HIV-1 as well as SIVmac239 and is observed in non-lymphoid cell lines of simian and murine origins. Two separate regions of HIV-1SF2 Nef are critical for the associated kinase activity. One domain overlaps with a central highly conserved region found in all primate lentivirus nef genes and has been provisionally mapped to amino acids 45-127. Because membrane localization of Nef is important for the associated cellular kinase activity, the second domain represents a membrane targeting signal. Moreover, point mutations within the central region that abrogate the Nef-associated kinase activity in HIV-1SF2 Nef have the same effect when introduced into SIVmac239open Nef.
Highlights
From the Cancer Research Institute, Department of Medicine, and §Howard Hughes Medical Institute, University of California, San Francisco, California 94143-0128
The ability of Nef to associate with the kinase activity is conserved among several alleles of HIV-I as well as SIVmac239 and is observed in non-lymphoid cell lines of simian and murine origins
It is conserved among all primate lentiviruses, i.e. HIV-l, HIV-2, and simian immunodeficiency virus (SIV) [2, 3]
Summary
Vol 270, No 25, Issue of June 23, pp. 15307~15314, 1995 Printed in U.S.A. A Conserved Domain and Membrane Targeting of Nef from HIV and SIV Are Required for Association with a Cellular Serine Kinase Activity*. We found Nef expressed either as a CDS-Nef fusion protein or as a native protein in virally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. We demonstrated that Nef expressed either as a CD8-Nef fusion protein or native Nef itself interacted with a serine kinase in human T-Iymphocytes [47] This kinase was found to phosphorylate proteins of and 72 kDa that coimmunoprecipitated with Nef. In the present study, the regions of Nef that are important for these interactions with the kinase activity were investigated.
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