RESUMO
The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734-736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.
Assuntos
Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Endocitose , Produtos do Gene env/genética , Macaca mulatta/metabolismo , Macaca nemestrina , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismoRESUMO
A chimeric vesicular stomatitis virus with the glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, is a potent viral vaccine vector that overcomes several of the limitations of wild-type VSV. Here, we evaluated the potential of VSV-GP as an HIV vaccine vector. We introduced genes for different variants of the HIV-1 envelope protein Env, i.e., secreted or membrane-anchored, intact or mutated furin cleavage site or different C-termini, into the genome of VSV-GP. We found that the addition of the Env antigen did not attenuate VSV-GP replication. All HIV-1 Env variants were expressed in VSV-GP infected cells and some were incorporated very efficiently into VSV-GP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV-1 such as MPER (membrane-proximal external region), CD4 binding site, V1V2 and V3 loop were present on the surface of VSV-GP-Env particles. Binding of quaternary antibodies indicated a trimeric structure of VSV-GP incorporated Env. We detected high HIV-1 antibody titers in mice and showed that vectors expressing membrane-anchored Env elicited higher antibody titers than vectors that secreted Envs. In rabbits, Tier 1A HIV-1 neutralizing antibodies were detectable after prime immunization and titers further increased after boosting with a second immunization. Taken together, VSV-GP-Env is a promising vector vaccine against HIV-1 infection since this vector permits incorporation of native monomeric and/or trimeric HIV-1 Env into a viral membrane.
Assuntos
Vacinas contra a AIDS/imunologia , Vetores Genéticos , Anticorpos Anti-HIV/sangue , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Feminino , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/genética , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Vírus da Estomatite Vesicular Indiana , Replicação ViralRESUMO
HIV-1 envelope (Env)-based vaccines have so far largely failed to induce antibodies that prevent HIV-1 infection. One factor proposed to limit the immunogenicity of cell-associated Env is its low level of expression on the cell surface, restricting accessibility to antibodies. Using a vaccinia prime/protein boost protocol in mice, we explored the immunologic effects of mutations in the Env cytoplasmic tail (CT) that increased surface expression, including partial truncation and ablation of a tyrosine-dependent endocytosis motif. After vaccinia primes, CT-modified Envs induced up to 7-fold higher gp120-specific IgG, and after gp120 protein boosts, they elicited up to 16-fold greater Tier-1 HIV-1 neutralizing antibody titers, although results were variable between isolates. These data indicate that the immunogenicity of HIV-1 Env in a prime/boost vaccine can be enhanced in a strain-dependent manner by CT mutations that increase Env surface expression, thus highlighting the importance of the prime in this vaccine format.
Assuntos
Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Vaccinia virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos , Feminino , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/administração & dosagem , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/genética , Humanos , Imunização Secundária , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
UNLABELLED: CD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for adaptive antiviral immune responses. Although CD4-independent variants of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have been described that can utilize the coreceptor CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and still can interact with CD4. We describe the derivation of a novel CD4-independent variant of pathogenic SIVmac239, termed iMac239, that was used to derive an infectious R5-tropic SIV lacking a CD4 binding site. Of the seven mutations that differentiate iMac239 from wild-type SIVmac239, a single change (D178G) in the V1/V2 region was sufficient to confer CD4 independence in cell-cell fusion assays, although other mutations were required for replication competence. Like other CD4-independent viruses, iMac239 was highly neutralization sensitive, although mutations were identified that could confer CD4-independent infection without increasing its neutralization sensitivity. Strikingly, iMac239 retained the ability to replicate in cell lines and primary cells even when its CD4 binding site had been ablated by deletion of a highly conserved aspartic acid at position 385, which, for HIV-1, plays a critical role in CD4 binding. iMac239, with and without the D385 deletion, exhibited an expanded host range in primary rhesus peripheral blood mononuclear cells that included CCR5(+) CD8(+) T cells. As the first non-CD4-tropic SIV, iMac239-ΔD385 will afford the opportunity to directly assess the in vivo role of CD4 targeting on pathogenesis and host immune responses. IMPORTANCE: CD4 tropism is an invariant feature of primate lentiviruses and likely plays a key role in pathogenesis by focusing viral infection onto cells that mediate adaptive immune responses and in protecting virions attached to cells from neutralizing antibodies. Although CD4-independent viruses are well described for HIV and SIV, these viruses characteristically retain their CD4 binding site and can engage CD4 if available. We derived a novel CD4-independent, CCR5-tropic variant of the pathogenic molecular clone SIVmac239, termed iMac239. The genetic determinants of iMac239's CD4 independence provide new insights into mechanisms that underlie this phenotype. This virus remained replication competent even after its CD4 binding site had been ablated by mutagenesis. As the first truly non-CD4-tropic SIV, lacking the capacity to interact with CD4, iMac239 will provide the unique opportunity to evaluate SIV pathogenesis and host immune responses in the absence of the immunomodulatory effects of CD4(+) T cell targeting and infection.
Assuntos
Antígenos CD4/metabolismo , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Tropismo Viral , Ligação Viral , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Antígenos CD4/imunologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Humanos , Leucócitos Mononucleares/virologia , Macaca mulatta , Mutagênese , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Receptores CXCR4/imunologia , Receptores CXCR4/metabolismo , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas do Envelope Viral/genética , Replicação Viral/genéticaRESUMO
UNLABELLED: Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques (Macaca mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4(+) T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques (Macaca nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4(+) T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of <15 to 50 RNA copies/ml. CD4(+) T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4(+) T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8(+) cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE: The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.
Assuntos
Motivos de Aminoácidos , Linfócitos T CD4-Positivos/imunologia , Imunidade nas Mucosas , Macaca nemestrina/virologia , Deleção de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Progressão da Doença , Feminino , Expressão Gênica , Intestinos/imunologia , Intestinos/virologia , Macaca mulatta/virologia , Masculino , Dados de Sequência Molecular , Mucosa/imunologia , Mucosa/virologia , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Especificidade da Espécie , Proteínas do Envelope Viral/deficiência , Proteínas do Envelope Viral/genética , Carga Viral/genética , Carga Viral/imunologia , Viremia/imunologia , Viremia/patologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
UNLABELLED: Chimeric simian immunodeficiency virus (SIV)/human immunodeficiency virus (HIV) (SHIV) infection of macaques is commonly used to model HIV type 1 (HIV-1) transmission and pathogenesis in humans. Despite the fact that SHIVs encode SIV antagonists of the known macaque host restriction factors, these viruses require additional adaptation for replication in macaques to establish a persistent infection. Additional adaptation may be required in part because macaque CD4 (mCD4) is a suboptimal receptor for most HIV-1 envelope glycoprotein (Env) variants. This requirement raises the possibility that adaptation of HIV-1 Env to the macaque host leads to selection of variants that lack important biological and antigenic properties of the viruses responsible for the HIV-1 pandemic in humans. Here, we investigated whether this adaptation process leads to changes in the antigenicity and structure of HIV-1 Env. For this purpose, we examined how two independent mutations that enhance mCD4-mediated entry, A204E and G312V, impact antibody recognition in the context of seven different parental HIV-1 Env proteins from diverse subtypes. We also examined HIV-1 Env variants from three SHIVs that had been adapted for increased replication in macaques. Our results indicate that these different macaque-adapted variants had features in common, including resistance to antibodies directed to quaternary epitopes and sensitivity to antibodies directed to epitopes in the variable domains (V2 and V3) that are buried in the parental, unadapted Env proteins. Collectively, these findings suggest that adaptation to mCD4 results in conformational changes that expose epitopes in the variable domains and disrupt quaternary epitopes in the native Env trimer. IMPORTANCE: These findings indicate the antigenic consequences of adapting HIV-1 Env to mCD4. They also suggest that to best mimic HIV-1 infection in humans when using the SHIV/macaque model, HIV-1 Env proteins should be identified that use mCD4 as a functional receptor and preserve quaternary epitopes characteristic of HIV-1 Env.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , HIV-1/imunologia , HIV-1/fisiologia , Mutação de Sentido Incorreto , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Adaptação Biológica , Animais , Antígenos CD4/metabolismo , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Humanos , Macaca , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Disruption of the conserved motif GYxxØ in the simian immunodeficiency virus (SIV) SIVmac239 envelope (Env) cytoplasmic tail resulted in a virus (ΔGY) that exhibited a high plasma peak but uniquely failed to acutely deplete mucosal CD4(+) T cells. Here, we show that ΔGY containing a flanking S727P mutation that was acquired in ΔGY-infected macaques reacquired the ability to rapidly deplete CD4(+) T cells in lamina propria. This suggests that the GYxxØ motif and S727P each contribute to SIV's targeting to mucosal tissues.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Mucosa/imunologia , Mutação de Sentido Incorreto , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Motivos de Aminoácidos , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Produtos do Gene env/química , Macaca , Masculino , Mucosa/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/metabolismoRESUMO
Food animal production systems have become more consolidated and integrated, producing large, concentrated animal populations and significant amounts of fecal waste. Increasing use of manure and litter as a more "natural" and affordable source of fertilizer may be contributing to contamination of fruits and vegetables with foodborne pathogens. In addition, human and animal manure have been identified as a significant source of antibiotic resistance genes thereby serving as a disseminator of resistance to soil and waterways. Therefore, identifying methods to remediate human and animal waste is critical in developing strategies to improve food safety and minimize the dissemination of antibiotic resistant bacteria. In this study, we sought to determine whether withdrawing antibiotic growth promoters or using alternatives to antibiotics would reduce the abundance of antibiotic resistance genes or prevalence of pathogens in poultry litter. Terminal restriction fragment length polymorphism (T-RFLP) paired with high throughput sequencing was used to evaluate the bacterial community composition of litter from broiler chickens that were treated with streptogramin growth-promoting antibiotics, probiotics, or prebiotics. The prevalence of resistance genes and pathogens was determined from sequencing results or PCR screens of litter community DNA. Streptogramin antibiotic usage did not elicit statistically significant differences in Shannon diversity indices or correlation coefficients among the flocks. However, T-RFLP revealed that there were inter-farm differences in the litter composition that was independent of antibiotic usage. The litter from all farms, regardless of antibiotic usage, contained streptogramin resistance genes (vatA, vatB, and vatE), macrolide-lincosamide-streptogramin B resistance genes (ermA and ermB), the tetracycline resistance gene tetM and class 1 integrons. There was inter-farm variability in the distribution of vatA and vatE with no statistically significant differences with regards to usage. Bacterial diversity was higher in litter when probiotics or prebiotics were administered to flocks but as the litter aged, diversity decreased. No statistically significant differences were detected in the abundance of class 1 integrons where 3%-5% of the community was estimated to harbor a copy. Abundance of pathogenic Clostridium species increased in aging litter despite the treatment while the abundance of tetracycline-resistant coliforms was unaffected by treatment. However some treatments decreased the prevalence of Salmonella. These findings suggest that withdrawing antibiotics or administering alternatives to antibiotics can change the litter bacterial community and reduce the prevalence of some pathogenic bacteria, but may not immediately impact the prevalence of antibiotic resistance.
Assuntos
Bactérias/efeitos dos fármacos , Bambermicinas/farmacologia , Galinhas , Probióticos/farmacologia , Virginiamicina/farmacologia , Animais , Antibacterianos/farmacologia , Bactérias/classificação , Bactérias/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Microbiologia Ambiental , Pisos e Cobertura de Pisos , Abrigo para Animais , RNA Ribossômico 16S/genéticaRESUMO
A hallmark of pathogenic simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections is the rapid and near-complete depletion of mucosal CD4(+) T lymphocytes from the gastrointestinal tract. Loss of these cells and disruption of epithelial barrier function are associated with microbial translocation, which has been proposed to drive chronic systemic immune activation and disease progression. Here, we evaluate in rhesus macaques a novel attenuated variant of pathogenic SIVmac239, termed ΔGY, which contains a deletion of a Tyr and a proximal Gly from a highly conserved YxxØ trafficking motif in the envelope cytoplasmic tail. Compared to SIVmac239, ΔGY established a comparable acute peak of viremia but only transiently infected lamina propria and caused little or no acute depletion of mucosal CD4(+) T cells and no detectable microbial translocation. Nonetheless, these animals developed T-cell activation and declining peripheral blood CD4(+) T cells and ultimately progressed with clinical or pathological features of AIDS. ΔGY-infected animals also showed no infection of macrophages or central nervous system tissues even in late-stage disease. Although the ΔGY mutation persisted, novel mutations evolved, including the formation of new YxxØ motifs in two of four animals. These findings indicate that disruption of this trafficking motif by the ΔGY mutation leads to a striking alteration in anatomic distribution of virus with sparing of lamina propria and a lack of microbial translocation. Because these animals exhibited wild-type levels of acute viremia and immune activation, our findings indicate that these pathological events are dissociable and that immune activation unrelated to gut damage can be sufficient for the development of AIDS.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas do Envelope Viral/metabolismo , Fatores de Virulência/metabolismo , Animais , Sistema Nervoso Central/virologia , Progressão da Doença , Feminino , Trato Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Macaca mulatta , Macrófagos/virologia , Masculino , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Transporte Proteico , RNA Viral/genética , Análise de Sequência de DNA , Deleção de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Proteínas do Envelope Viral/genética , Fatores de Virulência/genéticaRESUMO
HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Desoxirribonucleases/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Receptores CXCR4/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Proliferação de Células , Desoxirribonucleases/biossíntese , Desoxirribonucleases/genética , Modelos Animais de Doenças , Engenharia Genética , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/terapia , HIV-1/genética , HIV-1/metabolismo , Humanos , Macaca mulatta , Camundongos , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transplante Autólogo , Transplante Heterólogo , Internalização do VírusRESUMO
During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed DeltaV3-X4A.c5 and DeltaV3-X4B.c7, which took distinct molecular pathways for this change. The DeltaV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the DeltaV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to DeltaV3-X4A.c5, DeltaV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Receptores CXCR4/metabolismo , Receptores Virais/metabolismo , Tropismo Viral , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/química , Receptores CXCR4/genética , Receptores Virais/química , Receptores Virais/genética , Alinhamento de SequênciaRESUMO
We previously reported that a human immunodeficiency virus type 1 (HIV-1) clade B envelope protein with a severely truncated V3 loop regained function after passage in tissue culture. The adapted virus, termed TA1, retained the V3 truncation, was exquisitely sensitive to neutralization by the CD4 binding site monoclonal antibody b12 and by HIV-positive human sera, used CCR5 to enter cells, and was completely resistant to small molecule CCR5 antagonists. To examine the mechanistic basis for these properties, we singly and in combination introduced each of the 5 mutations from the adapted clone TA1 into the unadapted envelope. We found that single amino acid changes in the C3 region, the V3 loop, and in the fusion peptide were responsible for imparting near-normal levels of envelope function to TA1. T342A, which resulted in the loss of a highly conserved glycosylation site in C3, played the primary role. The adaptive amino acid changes had no impact on CCR5 antagonist resistance but made virus more sensitive to neutralization by antibodies to the CD4 binding site, modestly enhanced affinity for CD4, and made TA1 more responsive to CD4 binding. Specifically, TA1 was triggered by soluble CD4 more readily than the parental Env and, unlike the parental Env, could mediate entry on cells that express low levels of CD4. In contrast, TA1 interacted with CCR5 less efficiently and was highly sensitive to antibodies that bind to the CCR5 N terminus and ECL2. Therefore, enhanced utilization of CD4 is one mechanism by which HIV-1 can overcome mutations in the V3 region that negatively affect CCR5 interactions.
Assuntos
Fármacos Anti-HIV/imunologia , Antígenos CD4/imunologia , Proteína gp120 do Envelope de HIV , HIV-1/imunologia , Mutação , Receptores CCR5/imunologia , Adaptação Biológica/genética , Animais , Anticorpos Monoclonais/imunologia , Antagonistas dos Receptores CCR5 , Linhagem Celular , Cicloexanos/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/química , Humanos , Maraviroc , Receptores CCR5/genética , Proteínas Recombinantes/imunologia , Triazóis/metabolismoRESUMO
Like human immunodeficiency virus type 1 (HIV-1), most simian immunodeficiency virus (SIV) strains use CCR5 to establish infection. However, while HIV-1 can acquire the ability to use CXCR4, SIVs that utilize CXCR4 have rarely been reported. To explore possible barriers against SIV coreceptor switching, we derived an R5X4 variant, termed 239-ST1, from the R5 clone SIVmac239 by serially passaging virus in CD4(+) CXCR4(+) CCR5(-) SupT1 cells. A 239-ST1 env clone, designated 239-ST1.2-32, used CXCR4 and CCR5 in cell-cell fusion and reporter virus infection assays and conferred the ability for rapid, cytopathic infection of SupT1 cells to SIVmac239. Viral replication was inhibitable by the CXCR4-specific antagonist AMD3100, and replication was abrogated in a novel CXCR4(-) SupT1 line. Surprisingly, parental SIVmac239 exhibited low-level replication in SupT1 cells that was not observed in CXCR4(-) SupT1 cells. Only two mutations in the 239-ST1.2-32 Env, K47E in the C1 domain and L328W in the V3 loop, were required for CXCR4 use in cell-cell fusion assays, although two other V3 changes, N316K and I324M, improved CXCR4 use in infection assays. An Env cytoplasmic tail truncation, acquired during propagation of 239-ST1 in SupT1 cells, was not required. Compared with SIVmac239, 239-ST1.2-32 was more sensitive to neutralization by five of seven serum and plasma samples from SIVmac239-infected rhesus macaques and was approximately 50-fold more sensitive to soluble CD4. Thus, SIVmac239 can acquire the ability to use CXCR4 with high efficiency, but the changes required for this phenotype may be distinct from those for HIV-1 CXCR4 use. This finding, along with the increased neutralization sensitivity of this CXCR4-using SIV, suggests a mechanism that could select strongly against this phenotype in vivo.
Assuntos
HIV-1/metabolismo , Receptores CXCR4/genética , Vírus da Imunodeficiência Símia/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Humanos , Cinética , Luciferases/metabolismo , Macaca , Dados de Sequência Molecular , Fenótipo , Plasmídeos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The human immunodeficiency virus type 1 (HIV-1) V3 loop is essential for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. Using the dual-tropic virus HIV-1(R3A), we previously made an extensive panel of V3 deletions and identified subdomains within V3 that could differentially mediate R5 and X4 tropism. A deletion of residues 9 to 12 on the N-terminal side of the V3 stem ablated X4 tropism while leaving R5 tropism intact. This mutation also resulted in complete resistance to several small-molecule CCR5 inhibitors. Here, we extend these studies to further characterize a variant of this mutant, Delta9-12a, adapted for growth in CCR5(+) SupT1 cells. Studies using coreceptor chimeras, monoclonal antibodies directed against the CCR5 amino terminus (NT) and extracellular loops, and CCR5 point mutants revealed that, relative to parental R3A, R5-tropic Delta9-12a was more dependent on the CCR5 NT, a region that contacts the gp120 bridging sheet and V3 base. Neutralization sensitivity assays showed that, compared to parental R3A, Delta9-12a was more sensitive to monoclonal antibodies b12, 4E10, and 2G12. Finally, cross-antagonism assays showed that Delta9-12a could use aplaviroc-bound CCR5 for entry. These studies indicate that increased dependence on the CCR5 NT represents a mechanism by which HIV envelopes acquire resistance to CCR5 antagonists and may have more general implications for mechanisms of drug resistance that arise in vivo. In addition, envelopes such as Delta9-12a may be useful for developing new entry inhibitors that target the interaction of gp120 and the CCR5 NT.
Assuntos
Benzoatos/farmacologia , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/genética , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Piperazinas/farmacologia , Compostos de Espiro/farmacologia , Internalização do Vírus/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Linhagem Celular , Dicetopiperazinas , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Ligação Proteica , Receptores CCR5/fisiologia , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
The human immunodeficiency virus type 1 (HIV-1) V3 loop is critical for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. The recent crystallographic resolution of V3 shows that its base is closely associated with the conserved coreceptor binding site on the gp120 core, whereas more distal regions protrude toward the cell surface, likely mediating interactions with coreceptor extracellular loops. However, these V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity are poorly understood. Using the dual-tropic virus HIV-1(R3A), which uses both CCR5 and CXCR4, we sought to identify subdomains within V3 that selectively mediate R5 or X4 tropism. An extensive panel of V3 mutants was evaluated for effects on tropism and sensitivity to coreceptor antagonists. Mutations on either side of the V3 base (residues 3 to 8 and 26 to 33) ablated R5 tropism and made the resulting X4-tropic Envs more sensitive to the CXCR4 inhibitor AMD3100. When mutations were introduced within the V3 stem, only a deletion of residues 9 to 12 on the N-terminal side ablated X4 tropism. Remarkably, this R5-tropic Delta9-12 mutant was completely resistant to several small-molecule inhibitors of CCR5. Envs with mutations in the V3 crown (residues 13 to 20) remained dual tropic. Similar observations were made for a second dual-tropic isolate, HIV-1(89.6). These findings suggest that V3 subdomains can be identified that differentially affect R5 and X4 tropism and modulate sensitivity to CCR5 and CXCR4 inhibitors. These studies provide a novel approach for probing V3-coreceptor interactions and mechanisms by which these interactions can be inhibited.
Assuntos
Proteína gp120 do Envelope de HIV/genética , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Internalização do Vírus , Linhagem Celular , Humanos , Mutagênese Sítio-Dirigida , Deleção de SequênciaRESUMO
The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.
Assuntos
Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Quimiocinas/imunologia , Receptores de HIV/antagonistas & inibidores , Sequência de Bases , Linhagem Celular , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/imunologia , HIV-1/química , HIV-1/genética , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Receptores CCR5/química , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Receptores de HIV/fisiologia , Replicação ViralRESUMO
Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.