Rhéaume E, Champagne P, Sekaly RP Clinical Research Institute of Montreal, Montreal, Quebec, Canada.
Although the exact function of the HIV-I Nef protein is still unknown, it is accepted that Nef causes a down-regulation of CD4 cell surface expression in human and mouse T-cells. One hypothesis is that Nef could augment CD4 degradation through dissociation of the CD4-p56lck complex following interaction of Nef with p56lck. To test this hypothesis, we used the sensitive in vivo yeast two-hybrid system. Constructions of CD4 cytoplasmic domain, full-length p56lck, p56lck's SH3 domain, full-length Nef, and Nef's first 148 a. a. were made as fusions to the DNA binding domain (BD) and activation domain (AD) of GAL4 in vectors pAS2 and pGAD424, respectively. Combinations of these plasmids and control plasmids were co-transformed in yeast strain SFY526 and interactions between tested proteins were assayed by liquid β gal assays using ONPG as substrate. Whereas negative controls gave β gal U values of 0.15 plus or minus
0.04, we obtained values of 1.04 plus or minus 0.06 and 1.5 plus or minus0.4 β gal U for the combinations of BD-CD4 with AD-p56lck and BD-p56lck with AD-p56lck, respectively. When we tested the combination of BD-p56lck with AD-Nef, we obtained a 13-gal U value of 2.3 plus or minus 0.4 compared with 0.18 plus or minus 0.02 for the BD-p561ck SH3 with AD-Nef combination. Thus, it appears that an interaction between Nef and p56lck is possible in vivo and that this interaction does not depend only on the p56lck's SH3 domain. Further work has been initiated to confirm this interaction in mammalian cells.
