RESUMEN
Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/genética , Antígenos CD4/genética , Catarrinos/genética , Catarrinos/virología , Variación Genética , VIH , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Virus de la Inmunodeficiencia de los Simios , Alelos , Animales , Antígenos CD4/química , Evolución Molecular , Productos del Gen env/química , Humanos , Unión Proteica , Dominios ProteicosRESUMEN
T-cell immunity is likely to play a role in protection against SARS-CoV-2 by helping generate neutralizing antibodies. We longitudinally studied CD4 T-cell responses to the M, N, and S structural proteins of SARS-CoV-2 in 26 convalescent individuals. Within the first two months following symptom onset, a majority of individuals (81%) mounted at least one CD4 T-cell response, and 48% of individuals mounted detectable SARS-CoV-2-specific circulating T follicular helper cells (cTfh, defined as CXCR5+PD1+ CD4 T cells). SARS-CoV-2-specific cTfh responses across all three protein specificities correlated with antibody neutralization with the strongest correlation observed for S protein-specific responses. When examined over time, cTfh responses, particularly to the M protein, increased in convalescence, and robust cTfh responses with magnitudes greater than 5% were detected at the second convalescent visit, a median of 38 days post-symptom onset. CD4 T-cell responses declined but persisted at low magnitudes three months and six months after symptom onset. These data deepen our understanding of antigen-specific cTfh responses in SARS-CoV-2 infection, suggesting that in addition to S protein, M and N protein-specific cTfh may also assist in the development of neutralizing antibodies and that cTfh response formation may be delayed in SARS-CoV-2 infection.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19/inmunología , COVID-19/virología , SARS-CoV-2/inmunología , Células T Auxiliares Foliculares/inmunología , Células T Auxiliares Foliculares/virología , Adulto , Anciano , Especificidad de Anticuerpos , Estudios de Casos y Controles , Proteínas de la Nucleocápside de Coronavirus/inmunología , Femenino , Interacciones Microbiota-Huesped/inmunología , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pandemias , Fosfoproteínas/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Factores de Tiempo , Proteínas de la Matriz Viral/inmunología , Adulto JovenRESUMEN
Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.
Asunto(s)
Antígenos CD4/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Virus de la Inmunodeficiencia de los Simios/genética , Proteínas del Envoltorio Viral/genética , Animales , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Evolución Molecular , Variación Genética/inmunología , VIH/genética , VIH/patogenicidad , Humanos , Pan troglodytes/genética , Pan troglodytes/inmunología , Polisacáridos/genética , Polisacáridos/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Not all persons recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection develop SARS-CoV-2-specific antibodies. We show that nonseroconversion is associated with younger age and higher reverse transcription PCR cycle threshold values and identify SARS-CoV-2 viral loads in the nasopharynx as a major correlate of the systemic antibody response.
Asunto(s)
COVID-19 , Formación de Anticuerpos , COVID-19/inmunología , Prueba Serológica para COVID-19 , Humanos , Nasofaringe , SARS-CoV-2 , SeroconversiónRESUMEN
Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Receptores CCR5/metabolismo , Receptores CXCR6/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Carga Viral , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Cercocebus atys , Macaca mulatta , Filogenia , Receptores CCR5/genética , Receptores CXCR6/genética , Homología de Secuencia , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Internalización del VirusRESUMEN
Sexual transmission of HIV-1 is an inefficient process, with only one or few variants of the donor quasispecies establishing the new infection. A critical, and as yet unresolved, question is whether the mucosal bottleneck selects for viruses with increased transmission fitness. Here, we characterized 300 limiting dilution-derived virus isolates from the plasma, and in some instances genital secretions, of eight HIV-1 donor and recipient pairs. Although there were no differences in the amount of virion-associated envelope glycoprotein, recipient isolates were on average threefold more infectious (P = 0.0001), replicated to 1.4-fold higher titers (P = 0.004), were released from infected cells 4.2-fold more efficiently (P < 0.00001), and were significantly more resistant to type I IFNs than the corresponding donor isolates. Remarkably, transmitted viruses exhibited 7.8-fold higher IFNα2 (P < 0.00001) and 39-fold higher IFNß (P < 0.00001) half-maximal inhibitory concentrations (IC50) than did donor isolates, and their odds of replicating in CD4+ T cells at the highest IFNα2 and IFNß doses were 35-fold (P < 0.00001) and 250-fold (P < 0.00001) greater, respectively. Interestingly, pretreatment of CD4+ T cells with IFNß, but not IFNα2, selected donor plasma isolates that exhibited a transmitted virus-like phenotype, and such viruses were also detected in the donor genital tract. These data indicate that transmitted viruses are phenotypically distinct, and that increased IFN resistance represents their most distinguishing property. Thus, the mucosal bottleneck selects for viruses that are able to replicate and spread efficiently in the face of a potent innate immune response.
Asunto(s)
Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , VIH-1/fisiología , Interferón Tipo I/inmunología , Femenino , Interacciones Huésped-Patógeno , Humanos , Masculino , Semen/virología , Ducha Vaginal , Virión , Replicación ViralRESUMEN
Simian immunodeficiency viruses (SIVs) use their Nef proteins to counteract the restriction factor tetherin. However, a deletion in human tetherin prevents antagonism by the Nef proteins of SIVcpz and SIVgor, which represent the ape precursors of human immunodeficiency virus type 1 (HIV-1). To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as a tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target a region adjacent to this deletion. In this study, we identified an unusual HIV-1 group O strain (RBF206) that evolved Vpu as an effective antagonist of human tetherin. While both RBF206 Vpu and Nef exert anti-tetherin activity in transient-transfection assays, mainly Vpu promotes RBF206 release in infected CD4+ T cells. Although mutations distinct from the adaptive changes observed in group M Vpus (M-Vpus) were critical for the acquisition of its anti-tetherin activity, RBF206 O-Vpu potently suppresses NF-κB activation and reduces CD4 cell surface expression. Interestingly, RBF206 Vpu counteracts tetherin in a largely species-independent manner, degrading both the long and short isoforms of human tetherin. Downmodulation of CD4, but not counteraction of tetherin, by RBF206 Vpu was dependent on the cellular ubiquitin ligase machinery. Our data present the first example of an HIV-1 group O Vpu that efficiently antagonizes human tetherin and suggest that counteraction by O-Nefs may be suboptimal.IMPORTANCE Previous studies showed that HIV-1 groups M and O evolved two alternative strategies to counteract the human ortholog of the restriction factor tetherin. While HIV-1 group M switched from Nef to Vpu due to a deletion in the cytoplasmic domain of human tetherin, HIV-1 group O, which lacks Vpu-mediated anti-tetherin activity, acquired a Nef protein that is able to target a region adjacent to the deletion. Here we report an unusual exception, identifying a strain of HIV-1 group O (RBF206) whose Vpu protein evolved an effective antagonism of human tetherin. Interestingly, the adaptive changes in RBF206 Vpu are distinct from those found in M-Vpus and mediate efficient counteraction of both the long and short isoforms of this restriction factor. Our results further illustrate the enormous flexibility of HIV-1 in counteracting human defense mechanisms.
Asunto(s)
VIH-1/inmunología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Liberación del Virus , Antígenos CD , Linfocitos T CD4-Positivos/virología , Línea Celular , Proteínas Ligadas a GPI/antagonistas & inhibidores , Humanos , FN-kappa B/antagonistas & inhibidoresRESUMEN
Antibodies recognizing conserved CD4-induced (CD4i) epitopes on human immunodeficiency virus type 1 (HIV-1) Env and able to mediate antibody-dependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV-1-infected cells to ADCC mediated by HIV-positive (HIV+) sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4 mimetics (CD4mc) that are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. However, HIV-1 developed additional mechanisms to avoid ADCC, including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 upregulation in response to alpha interferon (IFN-α) was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-ß and interleukin-27 (IL-27) also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals.IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIV+ sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-α treatment. Here we show that in addition to IFN-α, IFN-ß and IL-27 also affect Vpu-mediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD/genética , Antígenos CD4/inmunología , Epítopos/inmunología , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Evasión Inmune , Interferón beta/farmacología , Interleucinas/farmacología , Células Jurkat , Imitación Molecular , Regulación hacia Arriba , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
BACKGROUND: Simian immunodeficiency virus of chimpanzees (SIVcpz), the progenitor of human immunodeficiency virus type 1 (HIV-1), is associated with increased mortality and AIDS-like immunopathology in wild-living chimpanzees (Pan troglodytes). Surprisingly, however, similar findings have not been reported for chimpanzees experimentally infected with SIVcpz in captivity, raising questions about the intrinsic pathogenicity of this lentivirus. FINDINGS: Here, we report progressive immunodeficiency and clinical disease in a captive western chimpanzee (P. t. verus) infected twenty years ago by intrarectal inoculation with an SIVcpz strain (ANT) from a wild-caught eastern chimpanzee (P. t. schweinfurthii). With sustained plasma viral loads of 105 to 106 RNA copies/ml for the past 15 years, this chimpanzee developed CD4+ T cell depletion (220 cells/µl), thrombocytopenia (90,000 platelets/µl), and persistent soft tissue infections refractory to antibacterial therapy. Combination antiretroviral therapy consisting of emtricitabine (FTC), tenofovir disoproxil fumarate (TDF), and dolutegravir (DTG) decreased plasma viremia to undetectable levels (<200 copies/ml), improved CD4+ T cell counts (509 cell/µl), and resulted in the rapid resolution of all soft tissue infections. However, initial lack of adherence and/or differences in pharmacokinetics led to low plasma drug concentrations, which resulted in transient rebound viremia and the emergence of FTC resistance mutations (M184V/I) identical to those observed in HIV-1 infected humans. CONCLUSIONS: These data demonstrate that SIVcpz can cause immunodeficiency and other hallmarks of AIDS in captive chimpanzees, including P. t. verus apes that are not naturally infected with this virus. Moreover, SIVcpz-associated immunodeficiency can be effectively treated with antiretroviral therapy, although sufficiently high plasma concentrations must be maintained to prevent the emergence of drug resistance. These findings extend a growing body of evidence documenting the immunopathogenicity of SIVcpz and suggest that experimentally infected chimpanzees may benefit from clinical monitoring and therapeutic intervention.
Asunto(s)
Antirretrovirales/uso terapéutico , Enfermedades del Simio Antropoideo/tratamiento farmacológico , Enfermedades del Simio Antropoideo/virología , Pan troglodytes/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Animales , Antirretrovirales/administración & dosificación , Antirretrovirales/sangre , Terapia Antirretroviral Altamente Activa/efectos adversos , Recuento de Linfocito CD4 , Farmacorresistencia Viral , Masculino , Mutación , ARN Viral/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Carga Viral/efectos de los fármacosRESUMEN
Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee's main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4(+) T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses.
Asunto(s)
Citidina Desaminasa/genética , Infecciones por Lentivirus/genética , Pan troglodytes/inmunología , Pan troglodytes/virología , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Citidina Desaminasa/inmunología , Genes vif/genética , Haplorrinos , Lentivirus/genética , Infecciones por Lentivirus/inmunología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.
Asunto(s)
Desaminasa APOBEC-3G/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Polimorfismo Genético , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Desaminasa APOBEC-3G/genética , Alelos , Animales , Modelos Animales de Enfermedad , Virus de la Leucemia Murina de Friend/genética , Virus de la Leucemia Murina de Friend/fisiología , Humanos , Ratones , Ratones Transgénicos , Mutación , Replicación ViralRESUMEN
HIV-1 groups M and N emerged within the last century following two independent cross-species transmissions of SIVcpz from chimpanzees to humans. In contrast to pandemic group M strains, HIV-1 group N viruses are exceedingly rare, with only about a dozen infections identified, all but one in individuals from Cameroon. Poor adaptation to the human host may be responsible for this limited spread of HIV-1 group N in the human population. Here, we analyzed the function of Vpu proteins from seven group N strains from Cameroon, the place where this zoonosis originally emerged. We found that these N-Vpus acquired four amino acid substitutions (E15A, V19A and IV25/26LL) in their transmembrane domain (TMD) that allow efficient interaction with human tetherin. However, despite these adaptive changes, most N-Vpus still antagonize human tetherin only poorly and fail to down-modulate CD4, the natural killer (NK) cell ligand NTB-A as well as the lipid-antigen presenting protein CD1d. These functional deficiencies were mapped to amino acid changes in the cytoplasmic domain that disrupt putative adaptor protein binding sites and an otherwise highly conserved ßTrCP-binding DSGxxS motif. As a consequence, N-Vpus exhibited aberrant intracellular localization and/or failed to recruit the ubiquitin-ligase complex to induce tetherin degradation. The only exception was the Vpu of a group N strain recently discovered in France, but originally acquired in Togo, which contained intact cytoplasmic motifs and counteracted tetherin as effectively as the Vpus of pandemic HIV-1 M strains. These results indicate that HIV-1 group N Vpu is under strong host-specific selection pressure and that the acquisition of effective tetherin antagonism may lead to the emergence of viral variants with increased transmission fitness.
Asunto(s)
Antígenos CD/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Selección Genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Células Cultivadas , Citometría de Flujo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/metabolismo , VIH-1/patogenicidad , Humanos , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Homología de Secuencia de Aminoácido , Liberación del VirusRESUMEN
Broadly neutralizing antibodies targeting the V2 apex of the HIV-1 envelope trimer are among the most common specificities elicited in HIV-1-infected humans and simian-human immunodeficiency virus (SHIV)-infected macaques. To gain insight into the prevalent induction of these antibodies, we isolated and characterized 11 V2 apex-directed neutralizing antibody lineages from SHIV-infected rhesus macaques. Remarkably, all SHIV-induced V2 apex lineages were derived from reading frame two of the rhesus DH3-15*01 gene. Cryo-EM structures of envelope trimers in complex with antibodies from nine rhesus lineages revealed modes of recognition that mimicked three canonical human V2 apex-recognition modes. Notably, amino acids encoded by DH3-15*01 played divergent structural roles, inserting into a hole at the trimer apex, H-bonding to an exposed strand, or forming part of a loop scaffold. Overall, we identify a DH3-15*01-signature for rhesus V2 apex broadly neutralizing antibodies and show that highly selected genetic elements can play multiple roles in antigen recognition. Highlights: Isolated 11 V2 apex-targeted HIV-neutralizing lineages from 10 SHIV-infected Indian-origin rhesus macaquesCryo-EM structures of Fab-Env complexes for nine rhesus lineages reveal modes of recognition that mimic three modes of human V2 apex antibody recognitionAll SHIV-elicited V2 apex lineages, including two others previously published, derive from the same DH3-15*01 gene utilizing reading frame twoThe DH3-15*01 gene in reading frame two provides a necessary, but not sufficient, signature for V2 apex-directed broadly neutralizing antibodiesStructural roles played by DH3-15*01-encoded amino acids differed substantially in different lineages, even for those with the same recognition modePropose that the anionic, aromatic, and extended character of DH3-15*01 in reading frame two provides a selective advantage for V2 apex recognition compared to B cells derived from other D genes in the naïve rhesus repertoireDemonstrate that highly selected genetic elements can play multiple roles in antigen recognition, providing a structural means to enhance recognition diversity.
RESUMEN
BACKGROUND: The acquisition of effective Vpu-mediated anti-tetherin activity to promote virion release following transmission of SIVcpzPtt from central chimpanzees (Pan troglodytes troglodytes) to humans distinguishes pandemic HIV-1 group M strains from non-pandemic group N, O and P viruses and may have been a prerequisite for their global spread. Some functional motifs in the cytoplasmic region of HIV-1 M Vpus proposed to be important for anti-tetherin activity are more frequently found in the Vpu proteins of SIVcpzPtt than in those of SIVcpzPts infecting eastern chimpanzees (P. t. schweinfurthii), that have not been detected in humans, and SIVgor from gorillas, which is closely related to HIV-1 O and P. Thus, SIVcpzPtt strains may require fewer adaptive changes in Vpu than SIVcpzPts or SIVgor strains to counteract human tetherin. RESULTS: To examine whether SIVcpzPtt may only need changes in the transmembrane domain (TMD) of Vpu to acquire anti-tetherin activity, whereas SIVcpzPts and SIVgor may also require changes in the cytoplasmic region, we analyzed chimeras between the TMD of an HIV-1 M Vpu and the cytoplasmic domains of SIVcpzPtt (n = 2), SIVcpzPts (n = 2) and SIVgor (n = 2) Vpu proteins. Unexpectedly, all of these chimeras were capable of counteracting human tetherin to enhance virion release, irrespective of the presence or absence of the putative adaptor protein binding sites and the DSGxxS ß-TrCP binding motif reported to be critical for effective anti-tetherin activity of M Vpus. It was also surprising that in three of the six chimeras the gain of anti-tetherin function was associated with a loss of the CD4 degradation activity since this function was conserved among all parental HIV-1, SIVcpz and SIVgor Vpu proteins. CONCLUSIONS: Our results show that changes in the TMD of SIVcpzPtt, SIVcpzPts and SIVgor Vpus are sufficient to render them active against human tetherin. Thus, several previously described domains in the extracellular region of Vpu are not absolutely essential for tetherin antagonism but may be required for other Vpu functions.
Asunto(s)
VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Virus de la Inmunodeficiencia de los Simios/fisiología , Proteínas Reguladoras y Accesorias Virales/metabolismo , Liberación del Virus , Animales , Antígenos CD , Línea Celular , Proteínas Ligadas a GPI/antagonistas & inhibidores , Gorilla gorilla , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Pan troglodytes , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Virus de la Inmunodeficiencia de los Simios/genética , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX(n)L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX(n)L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.
Asunto(s)
Nucleocápside/química , Nucleocápside/metabolismo , Retroviridae/metabolismo , Liberación del Virus , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Productos del Gen gag/genética , Humanos , Virus de la Anemia Infecciosa Equina/genética , Virus de la Anemia Infecciosa Equina/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Retroviridae/clasificación , Retroviridae/genética , Alineación de Secuencia , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/metabolismo , Liberación del Virus/genéticaRESUMEN
Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-2(7312A), HIV-2(ST), and HIV-2(UC1). Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC(50)) NAb titers of 1.7 × 10(5), 2.8 × 10(4), and 3.3 × 10(4), respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-2(7312A) proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4(+) T cells and tested for neutralization sensitivity on human CD4(+) T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-2/inmunología , Secuencia de Aminoácidos , Formación de Anticuerpos , Línea Celular , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/inmunología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , VIH-1/inmunología , VIH-1/aislamiento & purificación , VIH-2/clasificación , VIH-2/genética , VIH-2/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Filogenia , Alineación de SecuenciaRESUMEN
Few studies have explored the role of neutralizing antibody (NAb) responses in controlling HIV-2 viremia and disease progression. Using a TZM-bl neutralization assay, we assessed heterologous and autologous NAb responses from a community cohort of HIV-2-infected individuals with a broad range of disease outcomes in rural Guinea-Bissau. All subjects (n = 40) displayed exceptionally high heterologous NAb titers (50% inhibitory plasma dilution or 50% inhibitory concentration [IC(50)], 1:7,000 to 1:1,000,000) against 5 novel primary HIV-2 envelopes and HIV-2 7312A, whereas ROD A and 3 primary envelopes were relatively resistant to neutralization. Most individuals also showed high autologous NAb against contemporaneous envelopes (78% of plasma-envelope combinations in 69 envelopes from 21 subjects), with IC(50)s above 1:10,000. No association between heterologous or autologous NAb titer and greater control of HIV-2 was found. A subset of envelopes was found to be more resistant to neutralization (by plasma and HIV-2 monoclonal antibodies). These envelopes were isolated from individuals with greater intrapatient sequence diversity and were associated with changes in potential N-linked glycosylation sites but not CD4 independence or CXCR4 use. Plasma collected from up to 15 years previously was able to potently neutralize recent autologous envelopes, suggesting a lack of escape from NAb and the persistence of neutralization-sensitive variants over time, despite significant NAb pressure. We conclude that despite the presence of broad and potent NAb responses in HIV-2-infected individuals, these are not the primary forces behind the dichotomous outcomes observed but reveal a limited capacity for adaptive selection and escape from host immunity in HIV-2 infection.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-2/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Formación de Anticuerpos , Línea Celular , Estudios de Cohortes , Femenino , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , VIH-1/aislamiento & purificación , VIH-1/fisiología , VIH-2/genética , VIH-2/aislamiento & purificación , VIH-2/fisiología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Alineación de Secuencia , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Recent studies have shown that natural infection by HIV-2 leads to the elicitation of high titers of broadly neutralizing antibodies (NAbs) against primary HIV-2 strains (T. I. de Silva, et al., J. Virol. 86:930-946, 2012; R. Kong, et al., J. Virol. 86:947-960, 2012; G. Ozkaya Sahin, et al., J. Virol. 86:961-971, 2012). Here, we describe the envelope (Env) binding and neutralization properties of 15 anti-HIV-2 human monoclonal antibodies (MAbs), 14 of which were newly generated from 9 chronically infected subjects. All 15 MAbs bound specifically to HIV-2 gp120 monomers and neutralized heterologous primary virus strains HIV-2(7312A) and HIV-2(ST). Ten of 15 MAbs neutralized a third heterologous primary virus strain, HIV-2(UC1). The median 50% inhibitory concentrations (IC(50)s) for these MAbs were surprisingly low, ranging from 0.007 to 0.028 µg/ml. Competitive Env binding studies revealed three MAb competition groups: CG-I, CG-II, and CG-III. Using peptide scanning, site-directed mutagenesis, chimeric Env constructions, and single-cycle virus neutralization assays, we mapped the epitope of CG-I antibodies to a linear region in variable loop 3 (V3), the epitope of CG-II antibodies to a conformational region centered on the carboxy terminus of V4, and the epitope(s) of CG-III antibodies to conformational regions associated with CD4- and coreceptor-binding sites. HIV-2 Env is thus highly immunogenic in vivo and elicits antibodies having diverse epitope specificities, high potency, and wide breadth. In contrast to the HIV-1 Env trimer, which is generally well shielded from antibody binding and neutralization, HIV-2 is surprisingly vulnerable to broadly reactive NAbs. The availability of 15 human MAbs targeting diverse HIV-2 Env epitopes can facilitate comparative studies of HIV/SIV Env structure, function, antigenicity, and immunogenicity.
Asunto(s)
Anticuerpos Monoclonales/química , Mapeo Epitopo/métodos , VIH-2/química , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/inmunología , Biotinilación , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/química , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Pruebas de Neutralización/métodos , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Chimpanzees in west central Africa (Pan troglodytes troglodytes) are endemically infected with simian immunodeficiency viruses (SIVcpzPtt) that have crossed the species barrier to humans and gorillas on at least five occasions, generating pandemic and nonpandemic forms of human immunodeficiency virus type 1 (HIV-1) as well as gorilla SIV (SIVgor). Chimpanzees in east Africa (Pan troglodytes schweinfurthii) are also infected with SIVcpz; however, their viruses (SIVcpzPts) have never been found in humans. To examine whether this is due to a paucity of natural infections, we used noninvasive methods to screen wild-living eastern chimpanzees in the Democratic Republic of the Congo (DRC), Uganda, and Rwanda. We also screened bonobos (Pan paniscus) in the DRC, a species not previously tested for SIV in the wild. Fecal samples (n = 3,108) were collected at 50 field sites, tested for species and subspecies origin, and screened for SIVcpz antibodies and nucleic acids. Of 2,565 samples from eastern chimpanzees, 323 were antibody positive and 92 contained viral RNA. The antibody-positive samples represented 76 individuals from 19 field sites, all sampled north of the Congo River in an area spanning 250,000 km(2). In this region, SIVcpzPts was common and widespread, with seven field sites exhibiting infection rates of 30% or greater. The overall prevalence of SIVcpzPts infection was 13.4% (95% confidence interval, 10.7% to 16.5%). In contrast, none of the 543 bonobo samples from six sites was antibody positive. All newly identified SIVcpzPts strains clustered in strict accordance to their subspecies origin; however, they exhibited considerable genetic diversity, especially in protein domains known to be under strong host selection pressure. Thus, the absence of SIVcpzPts zoonoses cannot be explained by an insufficient primate reservoir. Instead, greater adaptive hurdles may have prevented the successful colonization of humans by P. t. schweinfurthii viruses.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Linfocitos T CD4-Positivos/citología , República Democrática del Congo , Femenino , Variación Genética , Genoma , Geografía , Humanos , Funciones de Verosimilitud , Masculino , Datos de Secuencia Molecular , Pan paniscus , Pan troglodytes , Filogenia , Rwanda , Homología de Secuencia de Aminoácido , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Uganda , ViriónRESUMEN
Host restriction factors play key roles in innate antiviral defense, but it remains poorly understood which of them restricts HIV-1 in vivo. Here, we used single-cell transcriptomic analysis to identify host factors associated with HIV-1 control during acute infection by correlating host gene expression with viral RNA abundance within individual cells. Wide sequencing of cells from one participant with the highest plasma viral load revealed that intracellular viral RNA transcription correlates inversely with expression of the gene PTMA, which encodes prothymosin α. This association was genome-wide significant (Padjusted < 0.05) and was validated in 28 additional participants from Thailand and the Americas with HIV-1 CRF01_AE and subtype B infections, respectively. Overexpression of prothymosin α in vitro confirmed that this cellular factor inhibits HIV-1 transcription and infectious virus production. Our results identify prothymosin α as a host factor that restricts HIV-1 infection in vivo, which has implications for viral transmission and cure strategies.