HSN3 PROTEASOMAL SUBUNIT AS A TARGET FOR HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1 NEF PROTEIN

HIV-1 Nef protein is important for pathogenicity, but its biochemical function remains obscure. To clarify its role, a yeast two-hybrid syst em (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 b ehaved as bona fide Nef partner in ths control crosses. Nef-HsN3 inter action was confirmed by in vitro binding experiments. In particular, r ecombinant Nef was able to capture the HsN3 subunit from a natural pro teasome 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 t o aa 73-249, and the tract 219-249-reminiscent of SH3 domain N-termina l 3/5ths-was shown to be essential, though not sufficient. Attempts to purify a Nef-HsN3 complex from transfected COS7 cells were unsuccessf ul. However, Nef was found to markedly downregulate intracellular leve ls of both a coexpressed HsN3 and the endogenous simian homologue. The se results suggest that Nef, by binding to a subunit, might alter prot easome function in infected cells. (C) 1997 Academic Press.