STRUCTURAL, BIOCHEMICAL, AND BIOLOGICAL CHARACTERIZATION OF HIV NEF AND VPU

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Relatively high levels of the HIV-1 and HIV-2-negative effector factor (Nef) proteins were expressed in the insect cell line, Sf-9, with a baculovirus vector. Two forms of the baculovirus produced HIV-1 Nef proteins were purified to greater than 95% homogeneity by affinity chromatography on a Nef monoclonal antibody (S-897-55S) column. A bacterially-produced HIV-2 Nef protein was also produced at relatively high levels and purified to near homogeneity by gel filtration and ion exchange chromatography. A high-titered rabbit antiserum was developed against the full-length HIV-2 Nef protein, and was used to identify a 23kD Nef protein in HIV-2 (NIHZ)-infected H9 cells by radioimmunoprecipitation (RIP) assay. Both the HIV-1 Nef monoclonal antibody S-897-55S and the HIV-2 Nef rabbit anti-serum were used to study the in vitro oligomerization of Nef proteins. In addition, both the HIV-1 baculovirus and HIV-2 bacterial Nef proteins demonstrate autophosphorylation activities and are efficient substrates in vitro for protein kinase C activity. We have also shown that the HTLV-IIIB-infected H9 cell line expresses two distinct species of Nef proteins (p25 and p27), which are coded by two different nef genes expressed in different genetic variants of HIV-1. In addition, we are using a bacterially-expressed HIV-1 vpu protein to study its potential for binding calcium and/or binding calcium channel activator or inhibitor agents.