HsN3 proteasomal subunit as a target for human immunodeficiency virus type 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 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.
Rossi
F
F
Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, Pavia, I-27100, Italy.
Evstafieva
A
A
Pedrali-Noy
G
G
Gallina
A
A
Milanesi
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015693:Gene Products, nef / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D014162:Transfection; D014779:Virus Replication; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D009097:Multienzyme Complexes; D054311:nef Gene Products, Human Immunodeficiency Virus; D003546:Cysteine Endopeptidases; C108898:PSMB4 protein, human; D046988:Proteasome Endopeptidase Complex