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 there by the viral protease. Mutational analysis indicated that residues 54-60 in HIV-1 Nef were required for intravirion cleavage. When viruses were produced using T cell lines or primary lymphoblasts, these residues were also required for optimal viral infectivity. However, substitution 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 certain cleavage site residues to infectivity was conditional on the producer cell type. In particular, a mutant containing a deletion of residues 54-57 was phenotypically nef defective when produced using T cells (CEM, A2.01, or primary lymphoblasts) but was minimally impaired when produced from 293 or HeLa cells. This mutant was cleavage resistant, indicating that proteolytic processing of Nef was dispensable for infectivity enhancement when virions were assembled in certain non-T cells. Residues 54-61 of the cleavage site, including 54-57, were also required for Nef-mediated down-regulation of CD4. However, the surface expression of CD4 on HeLa cells in amounts comparable to that on the surface of primary T lymphoblasts did not create a producer cell environment in which residues 54-57 acquired greater virological importance. Furthermore, these residues were required for optimal infectivity even during virion assembly in T cells (A2. 01) that expressed a CD4 molecule that is unable to respond to Nef. These data suggested that in producer T cells, certain cleavage site residues (54-57) contribute to a Nef-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 Delta54-57 mutant separated genetically down-regulation 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 down-regulation.