INTRAVIRION GENERATION OF THE C-TERMINAL CORE DOMAIN OF HIV-1 NEF BY THE HIV-1 PROTEASE IS INSUFFICIENT TO ENHANCE VIRAL INFECTIVITY

Wild-type HIV-1 is more infectious than nef-deleted HIV-1 in both limi ting dilution and single-cycle infectivity assays. Moreover, Nef expre ssion from a separate plasmid in the virus-producing cells is capable of restoring the infectivity of genetically nef-deficient HIV-1. These observations indicate that the virion itself is altered by Nef expres sion to promote viral infectivity. Sucrose gradient-purified HIV-1 vir ions contain full-length Nef protein and its inclusion is dependent on N-terminal myristylation of Nef. As myristylation-defective mutants o i Nef do not enhance infectivity, incorporation of Nef into virions ma y mediate the enhanced infectivity. Studies with recombinant Nef have further shown that HIV-1 protease can cleave Nef into two polypeptides , a 20-kDa C-terminal core domain and a small N-terminal domain. Our a nalysis of purified HIV-I virions also showed a 20-kDa form of Nef. Th e generation of this 20-kDa form of Nef was inhibited by an HIV-1 prot ease inhibitor, and its C-terminal core domain identity was confirmed through epitope-tagging. Immunoblots of virions demonstrated that 60-8 0% of the incorporated Nef is cleaved by the HIV-1 protease. This find ing raised the possibility that the Nef core domain, which may no long er be tethered to the membrane due to absence of an N-terminal myristy l anchor, might mediate the enhanced infectivity. Therefore, a panel o f mutants surrounding the proteolytic cleavage site in Nef were analyz ed for effects on cleavage and enhancement of viral infectivity. Altho ugh some Nef mutants both failed to cleave and did not enhance viral i nfectivity, other mutants proved discordant in these functions. Specif ically, two mutants that contained point mutations in the N-terminal d omain cleaved normally, hence generating wild-type Nef core domain, ye t failed to enhance infectivity. Thus, although the majority of the Ne i protein in HIV-1 virions is cleaved by the viral protease into a 20- kDa C-terminal core domain, generation of this core domain of Nef appe ars insufficient to enhance HIV-1 infectivity. These findings suggest that protease cleavage of the Nef protein in virions is irrelevant for the infectivity function of Nef. (C) 1997 Academic Press.