VIROLOGICAL IMPORTANCE OF THE PROTEASE-CLEAVAGE SITE IN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF IS INDEPENDENT OF BOTH INTRAVIRION PROCESSING AND CD4 DOWN-REGULATION

The HIV-1 Nef protein is present within the virion and is processed th ere by the viral protease. Mutational analysis indicated that residues 54-60 in HIV-1 Nef were required for intravirion cleavage. When virus es were produced using T cell lines or primary lymphoblasts, these res idues were also required for optimal viral infectivity. However, subst itution of native Nef residues with those of a functional Gag cleavage site demonstrated that intravirion cleavage was insufficient for the virological function of this domain. Furthermore, the importance of ce rtain cleavage site residues to infectivity was conditional on the pro ducer cell type. In particular, a mutant containing a deletion of resi dues 54-57 was phenotypically nef defective when produced using T cell s (CEM, A2.01, or primary lymphoblasts) but was minimally impaired whe n produced from 293 or HeLa cells. This mutant was cleavage resistant, indicating that proteolytic processing of Nef was dispensable for inf ectivity enhancement when virions were assembled in certain non-T cell s. Residues 54-61 of the cleavage site, including 54-57 were also requ ired for Nef-mediated down-regulation of CD4. However, the surface exp ression of CD4 on HeLa cells in amounts comparable to that on the surf ace of primary T lymphoblasts did not create a producer cell environme nt in which residues 54-57 acquired greater virological importance. Fu rthermore, these residues were required for optimal infectivity even d uring virion assembly in T cells (A2.01) that expressed a CD4 molecule that is unable to respond to Nef. These data suggested that in produc er T cells, certain cleavage site residues (54-57) contribute to a Nei -mediated virological effect that is unlikely to be linked causally to CD4 down-regulation. Conversely, in the context of 293 cells as viral producers, the Delta 54-57 mutant separated genetically down-regulati on of CD4 (for which it was defective) from enhancement of infectivity (for which it was functional). Together, these data indicate that the virological function of the cleavage site domain is both independent of intravirion proteolytic processing of Nef and independent of CD4 do wn-regulation. (C) 1998 Academic Press.