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Background: CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively. Results: Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in AR5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bsdirected Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. Conclusion: Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals. [ABSTRACT FROM AUTHOR]

In efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef and other accessory genes. Although live attenuated HIV vaccines are unlikely to be developed due to a myriad of safety concerns, opportunities exist to better understand the correlates of immune protection against HIV infection by studying rare cohorts of long-term survivors infected with attenuated, nef-deleted HIV strains such as the Sydney blood bank cohort (SBBC). Here, we review studies of viral evolution, pathogenicity, and immune responses to HIV infection in SBBC members. The studies show that potent, broadly neutralizing anti-HIV antibodies and robust CD8+ T-cell responses to HIV infection were not necessary for long-term control of HIV infection in a subset of SBBC members, and were not sufficient to prevent HIV sequence evolution, augmentation of pathogenicity and eventual progression of HIV infection in another subset. However, a persistent T-helper proliferative response to HIV p24 antigen was associated with long-term control of infection. Together, these results underscore the importance of the host in the eventual outcome of infection. Thus, whilst generating an effective antibody and CD8+ T-cell response are an essential component of vaccines aimed at preventing primary HIV infection, T-helper responses may be important in the generation of an effective therapeutic vaccine aimed at blunting chronic HIV infection. [ABSTRACT FROM AUTHOR]

Background: The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with attenuated, nef-deleted variants of human immunodeficiency virus type 1 (HIV-1) acquired from a single source. Long-term prospective studies have demonstrated that the SBBC now comprises slow progressors (SP) as well as long-term nonprogressors (LTNP). Convergent evolution of nef sequences in SBBC SP and LTNP indicates the in vivo pathogenicity of HIV-1 in SBBC members is dictated by factors other than nef. To better understand mechanisms underlying the pathogenicity of nef-deleted HIV-1, we examined the phenotype and env sequence diversity of sequentially isolated viruses (n = 2) from 3 SBBC members. Results: The viruses characterized here were isolated from two SP spanning a three or six year period during progressive HIV-1 infection (subjects D36 and C98, respectively) and from a LTNP spanning a two year period during asymptomatic, nonprogressive infection (subject C18). Both isolates from D36 were R5X4 phenotype and, compared to control HIV-1 strains, replicated to low levels in peripheral blood mononuclear cells (PBMC). In contrast, both isolates from C98 and C18 were CCR5-restricted. Both viruses isolated from C98 replicated to barely detectable levels in PBMC, whereas both viruses isolated from C18 replicated to low levels, similar to those isolated from D36. Analysis of env by V1V2 and V3 heteroduplex tracking assay, V1V2 length polymorphisms, sequencing and phylogenetic analysis showed distinct intra- and inter-patient env evolution. Conclusion: Independent evolution of env despite convergent evolution of nef may contribute to the in vivo pathogenicity of nef-deleted HIV-1 in SBBC members, which may not necessarily be associated with changes in replication capacity or viral coreceptor specificity.

Background: The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with nef-deleted, attenuated strains of human immunodeficiency virus type 1 (HIV-1). Although the cohort members have experienced differing clinical courses and now comprise slow progressors (SP) as well as long-term nonprogressors (LTNP), longitudinal analysis of nef/long-terminal repeat (LTR) sequences demonstrated convergent nef/LTR sequence evolution in SBBC SP and LTNP. Thus, the in vivo pathogenicity of attenuated HIV-1 strains harboured by SBBC members is dictated by factors other than nef/LTR. Therefore, to determine whether defects in other viral genes contribute to attenuation of these HIV-1 strains, we characterized dominant HIV-1 rev alleles that persisted in 4 SBBC subjects; C18, C64, C98 and D36., Results: The ability of Rev derived from D36 and C64 to bind the Rev responsive element (RRE) in RNA binding assays was reduced by approximately 90% compared to Rev derived from HIV-1NL4-3, C18 or C98. D36 Rev also had a 50-60% reduction in ability to express Rev-dependent reporter constructs in mammalian cells. In contrast, C64 Rev had only marginally decreased Rev function despite attenuated RRE binding. In D36 and C64, attenuated RRE binding was associated with rare amino acid changes at 3 highly conserved residues; Gln to Pro at position 74 immediately N-terminal to the Rev activation domain, and Val to Leu and Ser to Pro at positions 104 and 106 at the Rev C-terminus, respectively. In D36, reduced Rev function was mapped to an unusual 13 amino acid extension at the Rev C-terminus., Conclusion: These findings provide new genetic and mechanistic insights important for Rev function, and suggest that Rev function, not Rev/RRE binding may be rate limiting for HIV-1 replication. In addition, attenuated rev alleles may contribute to viral attenuation and long-term survival of HIV-1 infection in a subset of SBBC members.