RESUMEN
BACKGROUND: HIV-1 DNA in blood monocytes is considered a viral source of various HIV-1 infected tissue macrophages, which is also known as "Trojan horse" hypothesis. However, whether these DNA can produce virions has been an open question for years, due to the inability of isolating high titer and infectious HIV-1 directly from monocytes. RESULTS: In this study, we demonstrated successful isolation of two strains of M-HIV-1 (1690 M and 1175 M) from two out of four study subjects, together with their in vivo controls, HIV-1 isolated from CD4+ T-cells (T-HIV-1), 1690 T and 1175 T. All M- and T- HIV-1 isolates were detected CCR5-tropic. Both M- HIV-1 exhibited higher levels of replication in monocyte-derived macrophages (MDM) than the two T- HIV-1. Consistent with our previous reports on the subject 1175 with late infection, compartmentalized env C2-V3-C3 sequences were identified between 1175 M and 1175 T. In contrast, 1690 M and 1690 T, which were isolated from subject 1690 with relatively earlier infection, showed homogenous env C2-V3-C3 sequences. However, multiple reverse transcriptase (RT) inhibitor resistance-associated variations were detected in the Gag-Pol region of 1690 M, but not of 1690 T. By further measuring HIV DNA intracellular copy numbers post-MDM infection, 1690 M was found to have significantly higher DNA synthesis efficiency than 1690 T in macrophages, indicating a higher RT activity, which was confirmed by AZT inhibitory assays. CONCLUSIONS: These results suggested that the M- and T- HIV-1 are compartmentalized in the two study subjects, respectively. Therefore, we demonstrated that under in vitro conditions, HIV-1 infected human monocytes can productively release live viruses while differentiating into macrophages.
Asunto(s)
VIH-1/aislamiento & purificación , VIH-1/fisiología , Monocitos/virología , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/virología , Activación Enzimática , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/clasificación , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Filogenia , Receptores CCR5/metabolismo , Receptores del VIH/metabolismo , Tropismo Viral/genética , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/química , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3' half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.
Asunto(s)
Variación Genética , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Sobrevivientes de VIH a Largo Plazo , Seronegatividad para VIH/inmunología , VIH-1/genética , VIH-1/patogenicidad , Parejas Sexuales , Linfocitos T Citotóxicos/inmunología , Citotoxicidad Inmunológica/inmunología , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/inmunología , Antígeno HLA-B27/metabolismo , Humanos , Activación de Linfocitos/inmunología , Masculino , Conducta Sexual , Sexo InseguroRESUMEN
HIV-1 viral protein R (Vpr) can induce cell cycle arrest and cell death, and may be beneficial in cancer therapy to suppress malignantly proliferative cell types, such as adult T-cell leukemia (ATL) cells. In this study, we examined the feasibility of employing the HIV-vpr gene, via targeted gene transfer, as a potential new therapy to kill ATL cells. We infected C8166 cells with a recombinant adenovirus carrying both vpr and GFP genes (rAd-vpr), as well as the vector control virus (rAd-vector). G(2)/M phase cell cycle arrest was observed in the rAd-vpr infected cells. Typical characteristics of apoptosis were detected in rAd-vpr infected cells, including sub-diploid peak exhibition in DNA content assay, the Hoechst 33342 accumulation, apoptotic body formation, mitochondrial membrane potential and plasma membrane integrity loss. The proteomic assay revealed apoptosis related protein changes, exhibiting the regulation of caspase-3 activity indicator proteins (vimentin and Rho GDP-dissociation inhibitor 2), mitochondrial protein (prohibitin) and other regulatory proteins. In addition, the up-regulation of anti-inflammatory redox protein, thioredoxin, was identified in the rAd-vpr infected group. Further supporting these findings, the increase of caspase 3&7 activity in the rAd-vpr infected group was observed. In conclusion, endogenous Vpr is able to kill HTLV-1 transformed C8166 cells, and may avoid the risks of inducing severe inflammatory responses through apoptosis-inducing and anti-inflammatory activities.
Asunto(s)
Apoptosis , VIH-1/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Proteoma/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Caspasas/metabolismo , Línea Celular Transformada , Membrana Celular/metabolismo , Electroforesis en Gel Bidimensional , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Datos de Secuencia Molecular , Péptidos/química , Transporte de Proteínas , Regulación hacia ArribaRESUMEN
BACKGROUND: Recent studies have shown that blood monocytes harbor human immunodeficiency virus type 1 (HIV-1) variants that are genotypically distinguishable from those in CD4(+) T cells. However, the biological function of monocyte-derived HIV-1 remains unclear. METHODS: Using pseudovirus assay, we analyzed the phenotype conferred by monocyte-derived HIV-1 envelopes from 8 patients. RESULTS: All pseudoviruses carrying monocyte-derived HIV-1 envelopes used CCR5; however, their use of additional coreceptors delineated 4 phenotypes in which viruses used (1) CCR5 only, (2) CCR5 and CXCR4, (3) CCR3 and CCR5, or (4) multiple coreceptors, including CCR1, CCR3, GPR15, CCR5, and CXCR4. More importantly, we observed 2 distinct cell tropism phenotypes for pseudoviruses carrying monocyte-derived envelopes: (1) monocyte-derived, macrophage-specific R5 (MDMS-R5) virus that, using CCR5 only, could infect monocyte-derived macrophages (MDMs) but not CD4(+) T cells and (2) dual tropic virus that infected both MDMs and primary CD4(+) T cells. We found blood monocytes harboring viruses with multiple phenotypes as early as 25 days before seroconversion and as late as 9 years after seroconversion. CONCLUSIONS: These data suggest that HIV-1 circulating in blood monocytes represents diverse HIV-1 with multiple phenotypes and that MDMS-R5 viruses may play an important role in infection with and persistence of HIV-1 within the monocyte/macrophage lineage.
Asunto(s)
VIH-1/clasificación , Macrófagos/virología , Monocitos/virología , Receptores CCR5/metabolismo , Adulto , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/virología , Femenino , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH , VIH-1/aislamiento & purificación , VIH-1/metabolismo , VIH-1/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fenotipo , Alineación de SecuenciaRESUMEN
The role of blood monocytes in HIV-1 infection is a relatively new field of interest. What happens to HIV-1 in monocytes and their relationship to CD4+ T cells before, during, and after suppressive antiretroviral therapy (ART) is largely unstudied. Here, considering that diversity is a good indicator of continued replication over time, we evaluated the effect of ART on HIV-1 in blood monocytes and CD4+ T cells by examining the diversity of HIV-1 from 4 infected patients who underwent and stopped therapy. We determined diversity and compartmentalization of HIV-1 between blood monocytes and CD4+ T cells in each patient in relationship to their ART regimens. Our data indicate that the rate of HIV-1 diversity increase in monocytes during therapy was significantly higher than in CD4+ T cells (P<0.05), suggesting that HIV-1 present in monocytes diversify more during therapy than in CD4+ T cells. Increased rates of HIV-1 compartmentalization between monocytes and CD4+ T cells while on therapy were also observed. These results suggest that ART inhibits HIV-1 replication in CD4+ T cells more than in blood monocytes and that better treatments to combat HIV-1 in monocytes/macrophages may be needed for a more complete suppression of HIV replication.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , VIH-1/aislamiento & purificación , Monocitos/virología , Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Progresión de la Enfermedad , Evolución Molecular , VIH-1/efectos de los fármacos , Humanos , Monocitos/efectos de los fármacos , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Relación Estructura-Actividad , Carga Viral , Replicación Viral/efectos de los fármacosRESUMEN
Candidate human immunodeficiency virus type 1 (HIV-1) vaccines designed to elicit T-cell immunity in HIV-1-uninfected persons are under investigation in phase I to III clinical trials. Little is known about how these vaccines impact the immunologic response postinfection in persons who break through despite vaccination. Here, we describe the first comprehensive characterization of HIV-specific T-cell immunity in vaccine study participants following breakthrough HIV-1 infection in comparison to 16 nonvaccinated subjects with primary HIV-1 infection. Whereas none of the 16 breakthrough infections possessed vaccine-induced HIV-1-specific T-cell responses preinfection, 85% of vaccinees and 86% of nonvaccinees with primary HIV-1 infection developed HIV-specific T-cell responses postinfection. Breakthrough subjects' T cells recognized 43 unique HIV-1 T-cell epitopes, of which 8 are newly described, and 25% were present in the vaccine. The frequencies of gamma interferon (IFN-gamma)-secreting cells recognizing epitopes within gene products that were and were not encoded by the vaccine were not different (P = 0.64), which suggests that responses were not anamnestic. Epitopes within Nef and Gag proteins were the most commonly recognized in both vaccinated and nonvaccinated infected subjects. One individual controlled viral replication without antiretroviral therapy and, notably, mounted a novel HIV-specific HLA-C14-restricted Gag LYNTVATL-specific T-cell response. Longitudinally, HIV-specific T cells in this individual were able to secrete IFN-gamma and tumor necrosis factor alpha, as well as proliferate and degranulate in response to their cognate antigenic peptides up to 5 years postinfection. In conclusion, a vaccinee's ability to mount an HIV-specific T-cell response postinfection is not compromised by previous immunization, since the CD8+ T-cell responses postinfection are similar to those seen in vaccine-naïve individuals. Finding an individual who is controlling infection highlights the importance of comprehensive studies of breakthrough infections in vaccine trials to determine whether host genetics/immune responses and/or viral characteristics are responsible for controlling viral replication.
Asunto(s)
Vacunas contra el SIDA/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Linfocitos T/inmunología , Linfocitos T/virología , Adulto , Secuencia de Aminoácidos , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Femenino , Infecciones por VIH/patología , Infecciones por VIH/terapia , VIH-1/química , Humanos , Inmunidad Innata/inmunología , Masculino , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
The lectins DC-SIGN and DC-SIGNR augment infection by human immunodeficiency virus (HIV), Ebolavirus (EBOV) and other pathogens. The neck domain of these proteins drives multimerization, which is believed to be required for efficient recognition of multivalent ligands. The neck domain of DC-SIGN consists of seven sequence repeats with rare variations. In contrast, the DC-SIGNR neck domain is polymorphic and, in addition to the wild type (wt) allele with seven repeat units, allelic forms with five and six sequence repeats are frequently found. A potential association of the DC-SIGNR genotype and risk of HIV-1 infection is currently under debate. Therefore, we investigated if DC-SIGNR alleles with five and six repeat units exhibit defects in pathogen capture. Here, we show that wt DC-SIGNR and patient derived alleles with five and six repeats bind viral glycoproteins, augment viral infection and tetramerize with comparable efficiency. Moreover, coexpression of wt DC-SIGNR and alleles with five repeats did not decrease the interaction with pathogens compared to expression of each allele alone, suggesting that potential formation of hetero-oligomers does not appreciably reduce pathogen binding, at least under conditions of high expression. Thus, our results do not provide evidence for diminished pathogen capture by DC-SIGNR alleles with five and six repeat units. Albeit, we cannot exclude that subtle, but in vivo relevant differences remained undetected, our analysis suggests that indirect mechanisms could account for the association of polymorphisms in the DC-SIGNR neck region with reduced risk of HIV-1 infection.
Asunto(s)
Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Lectinas Tipo C/química , Lectinas Tipo C/genética , Polimorfismo Genético , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Proteínas del Envoltorio Viral/metabolismo , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Lectinas Tipo C/metabolismo , Lectinas Tipo C/fisiología , Estructura Terciaria de Proteína/genética , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Proteínas del Envoltorio Viral/genéticaRESUMEN
The repeat region of DC-SIGNR (CD209L) is polymorphic on the genomic level, and, in a separate study, we observed a correlation between the DC-SIGNR genotype and HIV-1 susceptibility during sexual contact. However, previous investigations using immunohistochemistry failed to detect membrane-bound DC-SIGNR on cells in the genital and rectal mucosa. We therefore explored the presence of DC-SIGNR in these compartments with a more sensitive limiting dilution RT-PCR, which also allowed for quantification of alternatively spliced mRNA isoforms. DC-SIGN (CD209) and DC-SIGNR mRNA transcript isoforms were found in all 12 vaginal and two rectal biopsies obtained from 14 healthy individuals. For DC-SIGNR, we detected significantly more isoform than full-length transcripts (mean copy numbers/mug RNA: 602 vs 26; P=0.0009). Four mucosal samples lacked full-length DC-SIGNR transcripts entirely. Cloning and sequencing of DC-SIGNR mRNA in three additional individuals revealed a diverse repertoire of DC-SIGNR isoforms, many of which encoded for proteins predicted to be soluble and secreted. Indeed, in one vaginal sample, we detected only soluble isoforms. In conjunction with our prior observation that the DC-SIGNR genotype has an effect on HIV-1 transmission in vivo, these findings emphasize that DC-SIGNR, in addition to DC-SIGN, should be considered as a cofactor in sexual HIV-1 transmission. Soluble isoforms, in particular, may modulate the efficiency of viral transmission and dissemination.