title: Nef alleles from all major HIV-1 clades activate Src-family kinases and enhance HIV-1 replication in an inhibitor-sensitive manner.
abstract: The HIV-1 accessory factor Nef is essential for high-titer viral replication and AIDS progression. Nef function requires interaction with many host cell proteins, including specific members of the Src kinase family. Here we explored whether Src-family kinase activation is a conserved property of Nef alleles from a wide range of primary HIV-1 isolates and their sensitivity to selective pharmacological inhibitors. Representative Nef proteins from the major HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J and K strongly activated Hck and Lyn as well as c-Src to a lesser extent, demonstrating for the first time that Src-family kinase activation is a highly conserved property of primary M-group HIV-1 Nef isolates. Recently, we identified 4-amino substituted diphenylfuropyrimidines (DFPs) that selectively inhibit Nef-dependent activation of Src-family kinases as well as HIV replication. To determine whether DFP compounds exhibit broad-spectrum Nef-dependent antiretroviral activity against HIV-1, we first constructed chimeric forms of the HIV-1 strain NL4-3 expressing each of the primary Nef alleles. The infectivity and replication of these Nef chimeras was indistinguishable from that of wild-type virus in two distinct cell lines (U87MG astroglial cells and CEM-T4 lymphoblasts). Importantly, the 4-aminopropanol and 4-aminobutanol derivatives of DFP potently inhibited the replication of all chimeric forms of HIV-1 in both U87MG and CEM-T4 cells in a Nef-dependent manner. The antiretroviral effects of these compounds correlated with inhibition of Nef-dependent activation of endogenous Src-family kinases in the HIV-infected cells. Our results demonstrate that the activation of Hck, Lyn and c-Src by Nef is highly conserved among all major clades of HIV-1 and that selective targeting of this pathway uniformly inhibits HIV-1 replication.
authors.affiliation: Department of Infectious Disease and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Purushottam S
authors.initials: PS
authors.lastname: Narute
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019281:Dimerization; D015658:HIV Infections; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014779:Virus Replication; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D019380:Anti-HIV Agents; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases