Background:  The function of Vif in infected individuals remains poorly understood. Studies in vitro have demonstrated that Vif counteracts the APOBEC3G and 3F-induced cytidine deamination during reverse transcription, thus limiting the accumulation of deleterious G-to-A hypermutations. In addition, G-to-A mutations occur with a 5’ to 3’ graded frequency over the viral genome increasing toward nef until the polypurine tract after which they lessen. We have analyzed 298 nef sequences from a cohort of HIV-1-infected hemophiliacs for the presence of G-to-A hypermutations to highlight natural variations in Vif function in HIV-infected individuals.

Methods:  A total of 298 nef sequences were obtained from either plasma or peripheral blood mononuclear cells (PBMC) of 11 hemophiliacs (6 long-term non-progressors [LTNP] and 5 progressors) infected with HIV-1 before 1985. Sequences from 1995 and 1998 blood samples were analyzed using the program www.hiv.lanl.gov/HYPERMUT that compared each patient sequence to a patient-specific sequence to determine the frequency and context of G-to-A mutations. vif sequences were obtained by nested polymerase chain reaction (PCR) of PBMC DNA. A selected clone was expressed in VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR in non-permissive cells.

Results:  No hypermutation was found in 133 nef sequences from plasma virus of 11 patients, whereas 5 of 165 (3%) nef sequences from PBMC contained a total number of 116 G-to-A mutations (13.03% of G mutated). All 5 hypermutated sequences belonged to a single patient (LTNP-4) who has remained HIV-1 disease-free and has maintained CD4 T cell counts >500 cells/µL. The hypermutations clustered within the first 200 nucleotides of nef within the polypurine tract. The LTNP-4 vif gene was amplified from PBMC samples collected in 1995, 1998, and 2001. The analysis of 45 vif sequences showed a conservation of intact Vif. However, 15 amino acid changes persisted in LTNP-4 Vif as compared with NL4-3 Vif. The expression of Vif of LTNP-4 obtained in 1995 was significantly lower than NL4-3 Vif, however it could rescue the replication of a Vif-deleted virus in non-permissive cells.

Conclusions:  LTNP-4 is a single LTNP who is in a healthy condition after >20 years of infection. Her PBMC-associated HIV DNA showed hypermutated nef sequences suggestive of alterations of the Vif-APOBEC axis. This Vif indeed is characterized by mutations that render the viral protein either unstable or poorly expressed.