Doherty Institute - Research Publications
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ItemEvolution of DC-SIGN use revealed by fitness studies of R5 HIV-I variants emerging during AIDS progression(BMC, 2008-03-27)BACKGROUND: At early stages of infection CCR5 is the predominant HIV-1 coreceptor, but in approximately 50% of those infected CXCR4-using viruses emerge with disease progression. This coreceptor switch is correlated with an accelerated progression. However, those that maintain virus exclusively restricted to CCR5 (R5) also develop AIDS. We have previously reported that R5 variants in these "non-switch virus" patients evolve during disease progression towards a more replicative phenotype exhibiting altered CCR5 coreceptor interactions. DC-SIGN is a C-type lectin expressed by dendritic cells that HIV-1 may bind and utilize for enhanced infection of T cells in trans. To further explore the evolution of the R5 phenotype we analyzed sequential R5 isolates obtained before and after AIDS onset, i.e. at the chronic stage and during end-stage disease, with regard to efficiency of DC-SIGN use in trans-infections. RESULTS: Results from binding and trans-infection assays showed that R5 viruses emerging during end-stage AIDS disease displayed reduced ability to use DC-SIGN. To better understand viral determinants underlying altered DC-SIGN usage by R5 viruses, we cloned and sequenced the HIV-1 env gene. We found that end-stage R5 viruses lacked potential N-linked glycosylation sites (PNGS) in the gp120 V2 and V4 regions, which were present in the majority of the chronic stage R5 variants. One of these sites, amino acid position 160 (aa160) in the V2 region, also correlated with efficient use of DC-SIGN for binding and trans-infections. In fitness assays, where head-to-head competitions between chronic stage and AIDS R5 viruses were setup in parallel direct and DC-SIGN-mediated infections, results were further supported. Competitions revealed that R5 viruses obtained before AIDS onset, containing the V2 PNGS at aa160, were selected for in the trans-infection. Whereas, in agreement with our previous studies, the opposite was seen in direct target cell infections where end-stage viruses out-competed the chronic stage viruses. CONCLUSION: Results of our study suggest R5 virus variants with diverse fitness for direct and DC-SIGN-mediated trans-infections evolve within infected individuals at end-stage disease. In addition, our results point to the importance of a glycosylation site within the gp120 V2 region for efficient DC-SIGN use of HIV-1 R5 viruses.
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ItemAsn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS(BMC, 2007-12-12)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 A-R5 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 CD4bs-directed 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.
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ItemPhenotype and envelope gene diversity of nef-deleted HIV-1 isolated from long-term survivors infected from a single source(BMC, 2007-07-16)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.