Background:  Infection with HIV-1 encoding-defective nef variants may contribute to a relatively benign course of disease in a minority of long-term non-progressors (LTNP). We have examined the functions of nef alleles from 6 individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Of the 6 individuals, 4 have progressed to HIV disease (late progressors, LP), whereas the remaining 2 have maintained their LTNP status at least until 2003.

Methods:  The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells (PBMC) of all 6 individuals in 1995 and 1998. The major variant obtained in 1998 from plasma was cloned in the infectious molecular clone pNL4-3 in place of nef. These chimeric viruses were analyzed for infectivity and replication, CD4 and MHC class I antigen down-regulation in CEM-GFP cells and PBMC. Viral isolates were obtained by cocultivation of patients’ PBMC with allogenic T-cell blasts. Co-receptor use was determined by infecting U33 cells expressing CD4 and either CCR5 or CXCR4.

Results:  The major variant obtained in 1998 from plasma RNA from 5 of 6 individuals significantly reduced HIV infectivity and replication and impaired Nef-mediated CD4 but not MHC class I antigen down-modulation from the cell surface. The co-receptor usage of primary isolates obtained in 1995 and 1998 was next examined. Between 1995 and 1998, all but 1 viral isolate from LP were characterized by both CCR5 and CXCR4 use (R5X4 viruses); and 2 of 4 LP had switched their co-receptor use from R5 to R5X4. Sequencing of the gp120 Env V3 region confirmed an increase of net charge in the R5X4 isolates. In contrast, R5 viruses were isolated from the LTNP, except from 1 individual from whom no virus isolation, even after removal of CD8⁺ cells, was obtained.

Conclusions:  In individuals infected with nef-defective viruses, in vivo evolution of gp120 Env extending its co-receptor use to CXCR4 seems to be associated with HIV disease progression. This observation implies that the low levels of replication of nef-attenuated viruses is sufficient, and perhaps causatively involved, in Env-dependent viral evolution.