R. Welker et al., HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF PROTEIN IS INCORPORATED INTO VIRUS-PARTICLES AND SPECIFICALLY CLEAVED BY THE VIRAL PROTEINASE, Virology, 219(1), 1996, pp. 228-236
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.