RESUMO
Human immunodeficiency virus type 1 (HIV-1) nef undergoes adaptive evolution in the central nervous system (CNS), reflecting altered requirements for HIV-1 replication in macrophages/microglia and brain-specific immune selection pressures. The role of Nef in HIV-1 neurotropism and pathogenesis of HIV-associated dementia (HAD) is unclear. In this study, we characterized 82 nef alleles cloned from brain, cerebral spinal fluid, spinal cord, and blood/lymphoid tissue-derived HIV-1 isolates from seven subjects with HAD. CNS isolate-derived nef alleles were genetically compartmentalized and had reduced sequence diversity compared to those from lymphoid tissue isolates. Defective nef alleles predominated in a brain-derived isolate from one of the seven subjects (MACS2-br). The ability of Nef to down-modulate CD4 and MHC class 1 (MHC-1) was generally conserved among nef alleles from both CNS and lymphoid tissues. However, the potency of CD4 and MHC-1 down-modulation was variable, which was associated with sequence alterations known to influence these Nef functions. These results suggest that CD4 and MHC-1 down-modulations are highly conserved functions among nef alleles from CNS- and lymphoid tissue-derived HIV-1 isolates that may contribute to viral replication and escape from immune surveillance in the CNS.
Assuntos
Encéfalo/virologia , Antígenos CD4/metabolismo , Genes MHC Classe I , HIV-1/genética , Tecido Linfoide/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Complexo AIDS Demência/genética , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/virologia , Alelos , Sequência de Aminoácidos , Encéfalo/metabolismo , Linhagem Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/virologia , Regulação para Baixo , Genes nef , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Tecido Linfoide/metabolismo , Masculino , Dados de Sequência Molecular , Filogenia , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
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.
Assuntos
Produtos do Gene nef/genética , Produtos do Gene rev/genética , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Sobreviventes de Longo Prazo ao HIV , HIV-1/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Estudos de Coortes , Deleção de Genes , Expressão Gênica , Produtos do Gene rev/metabolismo , Genes env , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Ligação Proteica , Análise de Sequência de DNA , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood. We investigated mechanisms contributing to R5 envelope glycoprotein (Env)-mediated cellular apoptosis by constructing a panel of retroviral vectors engineered to co-express GFP and R5 Envs derived from two HIV-1-infected subjects spanning asymptomatic (Early, E-R5 Envs) to late stages of infection (Late, L-R5 Envs). The L-R5 Envs induced significantly more cellular apoptosis than E-R5 Envs, but only in Env-expressing (GFP-positive) cells, and only in cells where CD4 and CCR5 levels were limiting. Studies with fusion-defective Env mutants showed induction of apoptosis required membrane-fusing events. Our results provide evidence for an intracellular mechanism of R5 Env-induced apoptosis of CD4+ cells that requires membrane fusion. Furthermore, they contribute to a better understanding of mechanisms involved in CD4+ T-cell loss in subjects experiencing progressive R5 HIV-1 infection.