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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CP005120-11
Application #
3874624
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Division of Cancer Epidemiology and Genetics
Department
Type
DUNS #
City
State
Country
United States
Zip Code