HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF PROTEIN IS INCORPORATED INTO VIRUS-PARTICLES AND SPECIFICALLY CLEAVED BY THE VIRAL PROTEINASE

The Nef protein of primate immunodeficiency viruses is essential for e stablishing a highly productive pathogenic infection in vivo. In tissu e culture, Nef is not required for infection but enhances viral infect ivity. This effect is most pronounced in unstimulated primary lymphocy tes and occurs in the early phase of infection prior to viral gene exp ression. Since Nef expression does not lead to obvious changes in viru s composition, it was of interest to analyze whether Nef is incorporat ed into virus particles. Here, we show that Nef is specifically immuno precipitated from radioactively labeled human immunodeficiency virus t ype 1 (HIV-1)-infected cells and virus particle preparations. Quantita tive analysis revealed Nef to be incorporated on the order of 10% of r everse transcriptase incorporation, which corresponds to 5 to 10 molec ules of Nef per virion. In infected cells, Nef was detected as a full- length 27-kDa protein. In contrast, approximately 50% of particle-asso ciated Nef corresponded to an 18-kDa species which comigrated with the larger product after in vitro cleavage of purified HIV-1 Nef by the v iral proteinase. Nef cleavage in particle preparations was completely abolished by a specific inhibitor of HIV-1 proteinase. Most likely, Ne f is cleaved concomitantly with viral structural proteins on maturatio n of virus particles. This cleavage is likely to be functionally signi ficant because it dissociates the conserved core domain from the N-ter minal membrane attachment region. Our results suggest that the profoun d influence of Nef on establishing infection of unstimulated cells in tissue culture and in vivo is mediated by virion-associated Nef which functions in early infection before viral gene expression. (C) 1996 Ac ademic Press, Inc.