RESUMO
Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Intestino Delgado/imunologia , Receptores CCR/metabolismo , Transferência Adotiva , Animais , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Quimiocinas CC/metabolismo , Memória Imunológica , Intestino Delgado/virologia , Leucócitos Mononucleares/imunologia , Linfonodos/imunologia , Macaca mulatta , Transdução de Sinais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologiaRESUMO
The late (L) domain sequence used by mouse mammary tumor virus (MMTV) remains undefined. Similar to other L domain-containing proteins, MMTV p8 and p14NC proteins are monoubiquitinated, suggesting L domain function. Site-directed mutagenesis of p8, PLPPV, and p14NC, PLPPL, sequences in MMTV Gag revealed a requirement only for the PLPPV sequence in virion release in a position-dependent manner. Electron microscopy of a defective Gag mutant confirmed an L domain budding defect morphology. The equine infectious anemia virus (EIAV) YPDL core L domain sequence and PLPPV provided L domain function in reciprocal MMTV and EIAV Gag exchange mutants, respectively. Alanine scanning of the PLPPV sequence revealed a strict requirement for the valine residue but only minor requirements for any one of the other residues. Thus, PLPPV provides MMTV L domain function, representing a fourth type of retroviral L domain that enables MMTV Gag proteins to co-opt cellular budding pathways for release.
Assuntos
Motivos de Aminoácidos , Produtos do Gene gag/metabolismo , Vírus do Tumor Mamário do Camundongo/crescimento & desenvolvimento , Liberação de Vírus , Animais , Produtos do Gene gag/química , Produtos do Gene gag/genética , Células HEK293 , Humanos , Vírus do Tumor Mamário do Camundongo/química , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Microscopia EletrônicaRESUMO
For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag-Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag-Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.
Assuntos
Fármacos Anti-HIV/farmacologia , Benzamidas/farmacologia , HIV/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/efeitos dos fármacos , Acetilação , Sequência de Aminoácidos , Linhagem Celular , Cisteína/química , Proteínas de Fusão gag-pol/química , Proteínas de Fusão gag-pol/efeitos dos fármacos , Humanos , Lisina/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/químicaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0023703.].
RESUMO
HIV and SIV infection dynamics are commonly investigated by measuring plasma viral loads. However, this total viral load value represents the sum of many individual infection events, which are difficult to independently track using conventional sequencing approaches. To overcome this challenge, we generated a genetically tagged virus stock (SIVmac239M) with a 34-base genetic barcode inserted between the vpx and vpr accessory genes of the infectious molecular clone SIVmac239. Next-generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. In vitro infection of rhesus CD4+ T cells and in vivo infection of rhesus macaques revealed levels of viral replication of SIVmac239M comparable to parental SIVmac239. After intravenous inoculation of 2.2x105 infectious units of SIVmac239M, an average of 1,247 barcodes were identified during acute infection in 26 infected rhesus macaques. Of the barcodes identified in the stock, at least 85.6% actively replicated in at least one animal, and on average each barcode was found in 5 monkeys. Four infected animals were treated with combination antiretroviral therapy (cART) for 82 days starting on day 6 post-infection (study 1). Plasma viremia was reduced from >106 to <15 vRNA copies/mL by the time treatment was interrupted. Virus rapidly rebounded following treatment interruption and between 87 and 136 distinct clonotypes were detected in plasma at peak rebound viremia. This study confirmed that SIVmac239M viremia could be successfully curtailed with cART, and that upon cART discontinuation, rebounding viral variants could be identified and quantified. An additional 6 animals infected with SIVmac239M were treated with cART beginning on day 4 post-infection for 305, 374, or 482 days (study 2). Upon treatment interruption, between 4 and 8 distinct viral clonotypes were detected in each animal at peak rebound viremia. The relative proportions of the rebounding viral clonotypes, spanning a range of 5 logs, were largely preserved over time for each animal. The viral growth rate during recrudescence and the relative abundance of each rebounding clonotype were used to estimate the average frequency of reactivation per animal. Using these parameters, reactivation frequencies were calculated and ranged from 0.33-0.70 events per day, likely representing reactivation from long-lived latently infected cells. The use of SIVmac239M therefore provides a powerful tool to investigate SIV latency and the frequency of viral reactivation after treatment interruption.
Assuntos
Variação Genética , Genoma Viral/genética , Modelos Teóricos , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Animais , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Marcadores Genéticos/genética , Macaca mulatta , Masculino , Análise de Sequência de DNA , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/genética , Carga Viral , ViremiaRESUMO
Follicular helper CD4 T cells, TFH, residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8hCXCR5) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8hCXCR5 T cells were present throughout the follicles with some observed near infected TFH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication.IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, TFH, present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles, and those that do fail to robustly control viral replication in the TFH population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on TFH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus.
Assuntos
Linfócitos B/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Centro Germinativo/imunologia , Infecções por HIV/imunologia , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Animais , Linfócitos B/virologia , Linfócitos T CD8-Positivos/virologia , Engenharia Celular , Quimiotaxia , Centro Germinativo/citologia , Centro Germinativo/virologia , HIV-1/fisiologia , Humanos , Macaca mulatta , Receptores CXCR5/imunologia , Receptores de Retorno de Linfócitos/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Viremia , Replicação Viral/imunologiaRESUMO
AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4+ T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether an extensive, timely, and effective response could reduce the establishment of infection from a high-dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-simian immunodeficiency virus (anti-SIV) activity into rhesus macaques 3 days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single-genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific T-cell receptors (TCRs) had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (means of 4.0 versus 7.5 transmitted viral genotypes; P = 0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated means of the total number of founder viruses transmitted were 4.5 and 14.5 in the experimental and control groups, respectively (P = 0.021). Thus, a large antiviral T-cell response timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower-exposure doses in humans that typically transmit only a single virus. IMPORTANCE: The establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses induced by infection or vaccination are important contributors in limiting viral replication. However, in human immunodeficiency virus (HIV)/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus, the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans, where the virus exposure is considerably lower.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Vírus da Imunodeficiência Símia/imunologia , Transferência Adotiva/métodos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Humanos , Imunidade Celular/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vacinação/métodos , Replicação Viral/genéticaRESUMO
To study CD4(+)T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4(+)T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8(+)T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1ß, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4(+)T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4(+)effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4(+)T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4(+)T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4(+)T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model.
Assuntos
Linfócitos T CD4-Positivos/virologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Células Clonais , Produtos do Gene gag/imunologia , Macaca mulatta , Receptores de Antígenos de Linfócitos T/imunologia , Vírus da Imunodeficiência Símia/imunologia , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
BACKGROUND: The TRIM5α protein is a principal restriction factor that contributes to an HIV-1 replication block in rhesus macaque CD4+ T cells by preventing reverse transcription. HIV-1 restriction is induced in human CD4+ T cells by expression of rhesus TRIM5α as well as those of other old world monkeys. While TRIM5α restriction has been extensively studied in single-round infection assays, fewer studies have examined restriction after extended viral replication. RESULTS: To examine TRIM5α restriction of replication, we studied the ability of TRIM5α proteins from African green monkey (AgmTRIM5α) and gorilla (gorTRIM5α) to restrict HIV-1 and SIVmac239 replication. These xenogeneic TRIM5α genes were transduced into human Jurkat-CCR5 cells (JR5), which were then exposed to HIV-1 or SIVmac239. In our single-round infection assays, AgmTRIM5α showed a relatively modest 4- to 10-fold restriction of HIV-1 and SIVmac239, while gorTRIM5α produced a 2- and 3-fold restriction of HIV-1 and SIVmac239, respectively, consistent with the majority of previously published single-round studies. To assess the impact of these modest effects on infection, we tested restriction in replication systems initiated with either cell-free or cell-to-cell challenges. AgmTRIM5α powerfully restricted both HIV-1 and SIVmac239 replication 14 days after cell-free infection, with a ≥ 3-log effect. Moreover, expression of AgmTRIM5α restricted HIV-1 and SIVmac239 replication by 2-logs when co-cultured with infected JR5 cells for 12 days. In contrast, neither expression of gorTRIM5α nor rhesus TRIM5α induced significant resistance when co-cultured with infected cells. Follow up experiments showed that the observed differences between replication and infection were not due to assembly defects as xenogeneic TRIM5α expression had no effect on either virion production or specific infectivity. CONCLUSIONS: Our results indicate that AgmTRIM5α has a much greater effect on extended replication than on any single infection event, suggesting that AgmTRIM5α restriction acts cumulatively, building up over many rounds of replication. Furthermore, AgmTRIM5α was able to potently restrict both HIV-1 and SIV replication in a cell-to-cell infection challenge. Thus, AgmTRIM5α is unique among the TRIM5α species tested to date, being able to restrict even at the high multiplicities of infection presented by mixed culture with nonrestrictive infected cells.
Assuntos
Proteínas de Transporte/metabolismo , Chlorocebus aethiops/imunologia , HIV-1/imunologia , Vírus da Imunodeficiência Símia/imunologia , Integração Viral/efeitos dos fármacos , Animais , Gorilla gorilla/imunologia , HIV-1/fisiologia , Humanos , Células Jurkat , Vírus da Imunodeficiência Símia/fisiologiaRESUMO
Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and ß variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins.
Assuntos
Clonagem Molecular , Vetores Genéticos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Macaca mulatta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Transdução GenéticaRESUMO
UNLABELLED: The expression of xenogeneic TRIM5α proteins can restrict infection in various retrovirus/host cell pairings. Previously, we have shown that African green monkey TRIM5α (AgmTRIM5α) potently restricts both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus mac239 (SIV(mac239)) replication in a transformed human T-cell line (L. V. Coren, et al., Retrovirology 12:11, 2015, http://dx.doi.org/10.1186/s12977-015-0137-9). To assess AgmTRIM5α restriction in primary cells, we transduced AgmTRIM5α into primary rhesus macaque CD4 T cells and infected them with SIV(mac239). Experiments with T-cell clones revealed that AgmTRIM5α could reproducibly restrict SIV(mac239) replication, and that this restriction synergizes with an intrinsic resistance to infection present in some CD4 T-cell clones. AgmTRIM5α transduction of virus-specific CD4 T-cell clones increased and prolonged their ability to suppress SIV spread in CD4 target cells. This increased antiviral function was strongly linked to decreased viral replication in the AgmTRIM5α-expressing effectors, consistent with restriction preventing the virus-induced cytopathogenicity that disables effector function. Taken together, our data show that AgmTRIM5α restriction, although not absolute, reduces SIV replication in primary rhesus CD4 T cells which, in turn, increases their antiviral function. These results support prior in vivo data indicating that the contribution of virus-specific CD4 T-cell effectors to viral control is limited due to infection. IMPORTANCE: The potential of effector CD4 T cells to immunologically modulate SIV/HIV infection likely is limited by their susceptibility to infection and subsequent inactivation or elimination. Here, we show that AgmTRIM5α expression inhibits SIV spread in primary effector CD4 T cells in vitro. Importantly, protection of effector CD4 T cells by AgmTRIM5α markedly enhanced their antiviral function by delaying SIV infection, thereby extending their viability despite the presence of virus. Our in vitro data support prior in vivo HIV-1 studies suggesting that the antiviral CD4 effector response is impaired due to infection and subsequent cytopathogenicity. The ability of AgmTRIM5α expression to restrict SIV infection in primary rhesus effector CD4 T cells now opens an opportunity to use the SIV/rhesus macaque model to further elucidate the potential and scope of anti-AIDS virus effector CD4 T-cell function.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Proteínas de Transporte/metabolismo , Chlorocebus aethiops/genética , Macaca mulatta/imunologia , Vírus da Imunodeficiência Símia/imunologia , Replicação Viral/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte/genética , Citometria de Fluxo , Vetores Genéticos/genética , Retroviridae , Transdução Genética , Replicação Viral/genéticaRESUMO
BACKGROUND: The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8(+) T-cell control of SIV replication in CD4(+) T cells, we asked whether TCRs isolated from rhesus macaque CD8(+) T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8(+) T cells obtained from an uninfected/unvaccinated animal. PRINCIPAL FINDINGS: We transferred SIV-specific TCR genes isolated from rhesus macaque CD8(+) T-cell clones with varying abilities to suppress SIV replication in vitro into CD8(+) T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8(+) T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones. CONCLUSIONS: Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Genes Codificadores dos Receptores de Linfócitos T/genética , Macaca mulatta/imunologia , Vírus da Imunodeficiência Símia/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Transdução Genética , Replicação Viral/imunologia , Animais , Antígenos Virais , Técnicas de Cultura de Células , Células Clonais/imunologia , Humanos , Macaca mulatta/genéticaRESUMO
BACKGROUND: The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. RESULTS: Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. CONCLUSIONS: Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.
Assuntos
Capsídeo/metabolismo , Serina/genética , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Células Cultivadas , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação , Linfócitos T , Vírion/metabolismo , Vírion/ultraestrutura , Liberação de Vírus , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Studies using transformed human cell lines suggest that most SIV strains use CCR5 as co-receptor. Our analysis of primary rhesus macaque CD4(+) T-cell clones revealed marked differences in susceptibility to SIV(mac)239 infection. We investigated whether different levels of CCR5 expression account for clonal differences in SIV(mac)239 susceptibility. Macaque CD4(+) T-cells showed significant CCR5 downregulation 1-2days following CD3 mAb stimulation, which gradually recovered at resting state, 7-10days after activation. Exposure of clones to SIV(mac)239 during their CCR5(low) or CCR5(high) expression states revealed differences in SIV susceptibility independent of surface CCR5 levels. Furthermore, a CCR5 antagonist similarly reduced SIV(mac)239 infection of clones during their CCR5(low) or CCR5(high) expression states. Our data suggest a model where i) very low levels of CCR5 are sufficient for efficient SIV infection, ii) CCR5 levels above this threshold do not enhance infection, and iii) low level infection can occur in the absence of CCR5.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação para Baixo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores CCR5/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antagonistas dos Receptores CCR5 , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , DNA Viral/análise , Feminino , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene gag/metabolismo , Humanos , Macaca mulatta , Masculino , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CCR5/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologiaRESUMO
The zinc fingers of the HIV-1 nucleocapsid protein, NCp7, are prime targets for antiretroviral therapeutics. Here we show that S-acyl-2-mercaptobenzamide thioester (SAMT) chemotypes inhibit HIV by modifying the NCp7 region of Gag in infected cells, thereby blocking Gag processing and reducing infectivity. The thiol produced by SAMT reaction with NCp7 is acetylated by cellular enzymes to regenerate active SAMTs via a recycling mechanism unique among small-molecule inhibitors of HIV.
Assuntos
Fármacos Anti-HIV/farmacologia , Benzamidas/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Acetilação , Acilação , Fármacos Anti-HIV/química , Benzamidas/química , Genes gag/genética , Dados de Sequência Molecular , Bibliotecas de Moléculas Pequenas , Dedos de Zinco/efeitos dos fármacosRESUMO
Plasma viremia decreases coincident with the appearance of virus-specific CD8(+) T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8(+) T cell responses and transient increase in plasma viremia after depletion of CD8(+) T cells in SIV-infected monkeys strongly suggest a role for CD8(+) T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8(+) T cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8(+) T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9-, and Vif WY8-specific CD8(+) T cell clones to acutely infected rhesus macaques. The cells persisted in PBMCs between 4 and 9 d, but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs, where they persisted at high frequency for >6 wk. Although persisting cells in the lungs were Ag reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8(+) T cells demonstrated an escape mutation in this epitope <3 wk postinfection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8(+) T cells for evaluating immune control during acute infection and demonstrate that infused cells retain function and persist for at least 2 mo in specific tissues.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Memória Imunológica , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Transferência Adotiva , Animais , Sequência de Bases , Células Clonais , DNA Viral/genética , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Evasão da Resposta Imune/genética , Ativação Linfocitária/imunologia , Macaca mulatta , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/genética , Viremia/imunologiaRESUMO
CD8(+) T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8(+) T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4(+) T cells. We used a set of SIV(mac)239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I(high) or MHC class I(intermediate). However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I(low)) despite the observations that all CTL clones showed similar IFN-gamma responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Produtos do Gene nef/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica , Interferon gama/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , RNA Viral/metabolismo , Vírus da Imunodeficiência Símia/fisiologia , Replicação ViralRESUMO
Human immunodeficiency virus type 1 (HIV-1) Gag-RNA interactions are required for virus assembly. However, our prior study found that a defect in particle production exhibited by an HIV-1 proviral mutant with a severe deletion in the RNA-binding nucleocapsid (NC) region of Gag, NX, could be reversed by eliminating its protease activity. While our follow-up study indicated that a secondary RNA-binding site in Gag can also provide the required RNA-binding function, how protease activity inhibits NX virion production is still unclear. Therefore, we tested three possible mechanisms: NX virions are unstable and fall apart after budding; NX Gag assembly is slowed, allowing protease processing to start before particle formation; or the protease region within NX Gag-Pol becomes activated prematurely and processes the assembling Gag. We found that NX particles were as stable as wild-type virions. Furthermore, even a modest slowing of protease activity could rescue NX. Pulse-chase analysis revealed that the initial particle production by NC-deleted Gag was delayed compared to that of wild type Gag, but once started, the rate of production was similar, revealing a defect in the initiation of assembly. Wild-type Gag particle production was not eliminated or decreased in the presence of excess NX Gag-Pol, inconsistent with a premature activation of protease. Overall, these results indicate that the particle formation defect of NX is due to delayed initiation of assembly caused by the absence of NC in Gag, making it vulnerable to protease processing before budding can occur. Therefore, NC plays an important initiating role in Gag assembly.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Nucleocapsídeo/metabolismo , Processamento de Proteína Pós-Traducional , RNA Viral/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , HIV-1/genética , Humanos , Nucleocapsídeo/genética , Ligação Proteica , RNA Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
The presence of cellular proteins outside and inside retroviruses can indicate the roles they play in viral biology. However, experiments examining retroviruses can be complicated by the contamination of even highly purified virion preparations with nonviral particles (either microvesicles or exosomes). Two useful methods have been developed that can remove contaminating particles from virus stocks to produce highly pure virus preparations. One approach, the subtilisin digestion procedure, enzymatically removes the proteins outside the virions. While this method is well suited for the analysis of the interior proteins in the virions, it removes the extracellular domains of the integral membrane proteins on the virion. To preserve the proteins on the exterior of the virion for biochemical studies, a CD45 immunoaffinity depletion procedure that removes vesicles by capture with antibody-linked microbeads is employed. These methods allow for the isolation of highly purified virion preparations that are suitable for a wide variety of experiments, including the biochemical characterization of cellular proteins both on and in HIV virions, examination of virion/cell interactions, and imaging of virions.
Assuntos
Cromatografia de Afinidade/métodos , HIV-1/isolamento & purificação , Antígenos Comuns de Leucócito/metabolismo , Subtilisina/metabolismo , Vírion/isolamento & purificação , Humanos , MicroesferasRESUMO
Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P(2) was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P(2) to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P(2) interactions for particle release from cells.