HIV-1 Nef protein is important for pathogenicity, but its biochemical function remains obscure. To clarify its role, a yeast two-hybrid system (ths) screening was utilized to identify Nef cellular partners. Of 79 yeast clones harboring cDNAs for putative Nef binding proteins, 27 (34%) contained the coding region for HsN3 proteasomal subunit. HsN3 behaved as bona fide Nef partner in ths control crosses. Nef-HsN3 interaction was confirmed by in vitro binding experiments. In particular, recombinant Nef was able to capture the HsN3 subunit from a natural proteasome preparation. In Nef, the interacting region was mapped within aa 34-143, which span the structured portion of the protein, including the SH3-binding domain. In HsN3, Nef-binding portion was restricted to aa 73-249, and the tract 219-249-reminiscent of SH3 domain N-terminal 3/5ths-was shown to be essential, though not sufficient. Attempts to purify a Nef-HsN3 complex from transfected COS7 cells were unsuccessful. However, Nef was found to markedly downregulate intracellular levels of both a coexpressed HsN3 and the endogenous simian homologue. These results suggest that Nef, by binding to a subunit, might alter proteasome function in infected cells.
Copyright 1997 Academic Press.