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1.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2471-2474, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018507

RESUMO

HPV infection starts with the activation of the early promoter (EP) regulatory core and the replication of the viral particles to around 10-100 per cell at the beginning of the infection. For this reason, understanding the deterministic and stochastic role of the population number of viruses inside the cell is of pivotal importance to understand the regulation of the EP and the viral latency.The aim of this study is to extend a recently published minimal model of the EP transcriptional regulation in order to consider the effect of the viral population on gene regulation, to perform the bifurcation analysis and to understand the role of the stochasticity at the beginning of the infection.The bifurcation analysis showed how modeling the viral population number is pivotal to exhibit a bistable behavior, potentially linked to the viral latency. Moreover, the viral population number was identified as an important source of stochasticity, which is of paramount importance to drive the bistable switching mechanism in the first stages of infection.


Assuntos
Papillomaviridae , Infecções por Papillomavirus , Regulação Viral da Expressão Gênica , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/epidemiologia , Regiões Promotoras Genéticas , Latência Viral
2.
Rev Med Liege ; 75(9): 573-577, 2020 Sep.
Artigo em Francês | MEDLINE | ID: mdl-32909407

RESUMO

The human immunodeficiency virus (HIV), responsible for acquired immunodeficiency syndrome or AIDS, is a major public health problem. In Belgium, 2 to 3 new cases are diagnosed every day. Since the advent of combined antiretroviral treatments in 1996, the life expectancy and quality of life of infected patients have greatly improved. However, to date there is no cure for HIV. Individuals infected with HIV must remain on antiretroviral treatment for life. One of the reasons for the difficulty in finding a cure for HIV is that the virus can remain in a latent form, i.e. dormant, in some of the cells it infects. These latent reservoirs are not recognized by the immune system and can reactivate and thus restart the infection if the patient stops the treatment. These latent reservoirs are therefore a major obstacle to cure HIV and a great deal of research is being conducted by the scientific community to find an eradication strategy. In this article, we will present the different characteristics of these latent reservoirs and the different strategies put in place to identify and eliminate them.


Assuntos
Síndrome de Imunodeficiência Adquirida , Infecções por HIV , Bélgica , Humanos , Qualidade de Vida , Latência Viral
3.
PLoS Pathog ; 16(9): e1008834, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956422

RESUMO

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação Viral da Expressão Gênica , Inativação Gênica , HIV-1/fisiologia , Proteínas Repressoras , Fatores de Transcrição , Transcrição Genética , Latência Viral , Linfócitos T CD4-Positivos/virologia , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Humanos , Células Jurkat , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
4.
Nat Commun ; 11(1): 4148, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811834

RESUMO

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.


Assuntos
Desoxirribonucleases/genética , Dependovirus/genética , Infecções Oculares/terapia , Edição de Genes/métodos , Herpes Simples/terapia , Herpesvirus Humano 1/genética , Latência Viral/genética , Animais , Sistemas CRISPR-Cas/genética , Células Cultivadas , Chlorocebus aethiops , Infecções Oculares/genética , Infecções Oculares/virologia , Feminino , Células HEK293 , Herpes Simples/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/virologia , RNA-Seq , Análise de Célula Única , Gânglio Cervical Superior/metabolismo , Gânglio Cervical Superior/virologia , Células Vero
5.
PLoS Pathog ; 16(7): e1008701, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32735617

RESUMO

Gammaherpesviruses have evolved various strategies to take advantage of host cellular factors or signaling pathways to establish a lifelong latent infection. Like the human gammaherpesvirus Epstein-Barr virus, murine gammaherpesvirus 68 (MHV68) establishes and maintains latency in the memory B cells during infection of laboratory mice. We have previously shown that MHV68 can immortalize fetal liver-derived B cells that induce lymphomas when injected into immunodeficient mice. Here we identify interleukin 16 (IL16) as a most abundantly expressed cytokine in MHV68-immortalized B cells and show that MHV68 infection elevates IL16 expression. IL16 is not important for MHV68 lytic infection but plays a critical role in MHV68 reactivation from latency. IL16 deficiency increases MHV68 lytic gene expression in MHV68-immortalized B cells and enhances reactivation from splenic latency. Correlatively, IL16 deficiency increases the frequency of MHV68-infected plasma cells that can be attributed to enhanced MHV68 reactivation. Furthermore, similar to TPA-mediated lytic replication of Kaposi's sarcoma-associated herpesvirus, IL16 deficiency markedly induces Tyr705 STAT3 de-phosphorylation and elevates p21 expression, which can be counteracted by the tyrosine phosphatase inhibitor orthovanadate. Importantly, orthovanadate strongly blocks MHV68 lytic gene expression mediated by IL16 deficiency. These data demonstrate that virus-induced IL16 does not directly participate in MHV68 lytic replication, but rather inhibits virus reactivation to facilitate latent infection, in part through the STAT3-p21 axis.


Assuntos
Infecções por Herpesviridae/metabolismo , Interleucina-16/metabolismo , Infecções Tumorais por Vírus/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Linfócitos B/virologia , Infecções por Herpesviridae/imunologia , Interleucina-16/imunologia , Linfoma/virologia , Camundongos , Rhadinovirus/imunologia , Rhadinovirus/metabolismo
6.
PLoS Pathog ; 16(8): e1008679, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790802

RESUMO

Antiretroviral drugs that target various stages of the Human Immunodeficiency Virus (HIV) life cycle have been effective in curbing the AIDS epidemic. However, drug resistance, off-target effects of antiretroviral therapy (ART), and varying efficacy in prevention underscore the need to develop novel and alternative therapeutics. In this study, we investigated whether targeting the signaling molecule Sphingosine-1-phosphate (S1P) would inhibit HIV-1 infection and generation of the latent reservoir in primary CD4 T cells. We show that FTY720 (Fingolimod), an FDA-approved functional antagonist of S1P receptors, blocks cell-free and cell-to-cell transmission of HIV and consequently reduces detectable latent virus. Mechanistically, FTY720 impacts the HIV-1 life cycle at two levels. Firstly, FTY720 reduces the surface density of CD4, thereby inhibiting viral binding and fusion. Secondly, FTY720 decreases the phosphorylation of the innate HIV restriction factor SAMHD1 which is associated with reduced levels of total and integrated HIV, while reducing the expression of Cyclin D3. In conclusion, targeting the S1P pathway with FTY720 could be a novel strategy to inhibit HIV replication and reduce the seeding of the latent reservoir.


Assuntos
Cloridrato de Fingolimode/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/crescimento & desenvolvimento , Proteína 1 com Domínio SAM e Domínio HD/antagonistas & inibidores , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Linfócitos T/imunologia , Replicação Viral , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Lisofosfolipídeos/metabolismo , Fosforilação , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Linfócitos T/efeitos dos fármacos , Latência Viral
7.
Gac Med Mex ; 156(4): 328-333, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32831326

RESUMO

In the efforts to explain COVID-19 pathophysiology, studies are being carried out on the correspondence between the expression of SARS-CoV-2 cell receptors and viral sequences. ACE2, CD147 and TMPRSS2 receptors expression could indicate poorly explored potential infection targets. For the genomic analysis of SARS-CoV-2 receptors, using BioGPS information was decided, which is a portal that centralizes genetic annotation resources, in combination with that of The Human Protein Atlas, the largest portal of human transcriptome and proteome data. We also reviewed the most recent articles on the subject. RNA and viral receptor proteins expression was observed in numerous anatomical sites, which partially coincides with the information reported in the literature. High expression in testicular cells markedly stood out, and it would be therefore important ruling out whether this anatomical site is a SARS-CoV-2 reservoir; otherwise, germ cell damage, as it is observed in infections with other RNA viruses, should be determined.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Testículo/virologia , Basigina/genética , Infecções por Coronavirus/fisiopatologia , Regulação da Expressão Gênica , Humanos , Masculino , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral/fisiopatologia , Serina Endopeptidases/genética , Latência Viral
8.
Nature ; 585(7824): 261-267, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848246

RESUMO

Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected individuals (here termed 'elite controllers'), despite the presence of a replication-competent viral reservoir1. Inducing such an ability to spontaneously maintain undetectable plasma viraemia is a major objective of HIV-1 cure research, but the characteristics of proviral reservoirs in elite controllers remain to be determined. Here, using next-generation sequencing of near-full-length single HIV-1 genomes and corresponding chromosomal integration sites, we show that the proviral reservoirs of elite controllers frequently consist of oligoclonal to near-monoclonal clusters of intact proviral sequences. In contrast to individuals treated with long-term antiretroviral therapy, intact proviral sequences from elite controllers were integrated at highly distinct sites in the human genome and were preferentially located in centromeric satellite DNA or in Krüppel-associated box domain-containing zinc finger genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, the integration sites of intact proviral sequences from elite controllers showed an increased distance to transcriptional start sites and accessible chromatin of the host genome and were enriched in repressive chromatin marks. These data suggest that a distinct configuration of the proviral reservoir represents a structural correlate of natural viral control, and that the quality, rather than the quantity, of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, in one elite controller, we were unable to detect intact proviral sequences despite analysing more than 1.5 billion peripheral blood mononuclear cells, which raises the possibility that a sterilizing cure of HIV-1 infection, which has previously been observed only following allogeneic haematopoietic stem cell transplantation2,3, may be feasible in rare instances.


Assuntos
Inativação Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Heterocromatina/genética , Provírus/genética , Integração Viral/genética , Latência Viral/genética , Adulto , Idoso , Centrômero/genética , Cromossomos Humanos Par 19/genética , DNA Satélite/genética , Feminino , Genoma Viral/genética , Infecções por HIV/sangue , HIV-1/isolamento & purificação , Heterocromatina/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Provírus/isolamento & purificação , Proteínas Repressoras/genética , Sítio de Iniciação de Transcrição
9.
PLoS Pathog ; 16(8): e1008791, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841299

RESUMO

During antiretroviral therapy (ART) that suppresses HIV replication to below the limit-of-quantification, virions produced during ART can be detected at low frequencies in the plasma, termed residual viremia (RV). We hypothesized that a reservoir of HIV-infected cells actively produce and release virions during ART that are potentially infectious, and that following ART-interruption, these virions can complete full-cycles of replication and contribute to rebound viremia. Therefore, we studied the dynamics of RV sequence variants in 3 participants who initiated ART after ~3 years of infection and were ART-suppressed for >6 years prior to self-initiated ART-interruptions. Longitudinal RV C2V5env sequences were compared to sequences from pre-ART plasma, supernatants of quantitative viral outgrowth assays (QVOA) of cells collected during ART, post-ART-interruption plasma, and ART-re-suppression plasma. Identical, "putatively clonal," RV sequences comprised 8-84% of sequences from each timepoint. The majority of RV sequences were genetically similar to those from plasma collected just prior to ART-initiation, but as the duration of ART-suppression increased, an increasing proportion of RV variants were similar to sequences from earlier in infection. Identical sequences were detected in RV over a median of 3 years (range: 0.3-8.2) of ART-suppression. RV sequences were identical to pre-ART plasma viruses (5%), infectious viruses induced in QVOA (4%) and rebound viruses (5%) (total n = 21/154 (14%) across the 3 participants). RV sequences identical to ART-interruption "rebound" sequences were detected 0.1-7.4 years prior to ART-interruption. RV variant prevalence and persistence were not associated with detection of the variant among rebound sequences. Shortly after ART-re-suppression, variants that had been replicating during ART-interruptions were detected as RV (n = 5). These studies show a dynamic, virion-producing HIV reservoir that contributes to rekindling infection upon ART-interruption. The persistence of identical RV variants over years suggests that a subpopulation of HIV-infected clones frequently or continuously produce virions that may resist immune clearance; this suggests that cure strategies should target this active as well as latent reservoirs.


Assuntos
Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Plasma/virologia , Viremia/epidemiologia , Replicação Viral , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Incidência , Plasma/efeitos dos fármacos , Plasma/imunologia , Estudos Retrospectivos , Estados Unidos/epidemiologia , Carga Viral , Viremia/virologia , Latência Viral , Suspensão de Tratamento
10.
Proc Natl Acad Sci U S A ; 117(29): 17240-17248, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32632017

RESUMO

Probabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Superresolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein-the major viral transactivator protein-exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency.


Assuntos
Citomegalovirus/genética , Citomegalovirus/fisiologia , Proteínas Virais/metabolismo , Latência Viral/genética , Latência Viral/fisiologia , Evolução Biológica , Infecções por Citomegalovirus , Regulação Viral da Expressão Gênica , Humanos , Monócitos , Vírion/metabolismo , Replicação Viral
11.
PLoS Pathog ; 16(7): e1008683, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658923

RESUMO

Human herpesvirus 6B (HHV-6B) is a betaherpesvirus capable of integrating its genome into the telomeres of host chromosomes. Until now, the cellular and/or viral proteins facilitating HHV-6B integration have remained elusive. Here we show that a cellular protein, the promyelocytic leukemia protein (PML) that forms nuclear bodies (PML-NBs), associates with the HHV-6B immediate early 1 (IE1) protein at telomeres. We report enhanced levels of SUMOylated IE1 in the presence of PML and have identified a putative SUMO Interacting Motif (SIM) within IE1, essential for its nuclear distribution, overall SUMOylation and association with PML to nuclear bodies. Furthermore, using PML knockout cell lines we made the original observation that PML is required for efficient HHV-6B integration into host chromosomes. Taken together, we could demonstrate that PML-NBs are important for IE1 multiSUMOylation and that PML plays an important role in HHV-6B integration into chromosomes, a strategy developed by this virus to maintain its genome in its host over long periods of time.


Assuntos
Herpesvirus Humano 6/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Fosfoproteínas/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Infecções por Roseolovirus/metabolismo , Telômero/virologia , Linhagem Celular , Herpesvirus Humano 6/genética , Humanos , Infecções por Roseolovirus/genética , Sumoilação , Latência Viral/genética
12.
PLoS Pathog ; 16(7): e1008664, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678826

RESUMO

Establishing latent infection but retaining the capability to reactivate in certain circumstance is an ingenious tactic for retroviruses to persist in vivo while evading host immune surveillance. Many evidences indicate that Human T-cell leukemia virus type 1 (HTLV-1) is not completely silent in vivo. However, signals that trigger HTLV-1 latency-reactivation switching remain poorly understood. Here, we show that aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, plays a critical role in HTLV-1 plus-strand expression. Importantly, HTLV-1 reactivation could be tunably manipulated by modulating the level of AHR ligands. Mechanistically, activated AHR binds to HTLV-1 LTR dioxin response element (DRE) site (CACGCATAT) and drives plus-strand transcription. On the other hand, persistent activation of nuclear factor kappa B (NF-κB) pathway constitutes one key prerequisite for AHR overexpression in HTLV-1 infected T-cells, setting the stage for the advent of AHR signaling. Our findings suggest that HTLV-1 might achieve its reactivation in vivo when encountering environmental, dietary, microbial and metabolic cues that induce sufficient AHR signaling.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Linhagem Celular , Infecções por HTLV-I/metabolismo , Humanos , Linfócitos T/virologia
13.
PLoS Pathog ; 16(7): e1008644, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678836

RESUMO

The foamy viruses (FV) or spumaviruses are an ancient subfamily of retroviruses that infect a variety of vertebrates. FVs are endemic, but apparently apathogenic, in modern non-human primates. Like other retroviruses, FV replication is inhibited by type-I interferon (IFN). In a previously described screen of IFN-stimulated genes (ISGs), we identified the macaque PHD finger domain protein-11 (PHF11) as an inhibitor of prototype foamy virus (PFV) replication. Here, we show that human and macaque PHF11 inhibit the replication of multiple spumaviruses, but are inactive against several orthoretroviruses. Analysis of other mammalian PHF11 proteins revealed that antiviral activity is host species dependent. Using multiple reporter viruses and cell lines, we determined that PHF11 specifically inhibits a step in the replication cycle that is unique to FVs, namely basal transcription from the FV internal promoter (IP). In so doing, PHF11 prevents expression of the viral transactivator Tas and subsequent activation of the viral LTR promoter. These studies reveal a previously unreported inhibitory mechanism in mammalian cells, that targets a family of ancient viruses and may promote viral latency.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Infecções por Retroviridae/virologia , Spumavirus/fisiologia , Fatores de Transcrição/fisiologia , Latência Viral/fisiologia , Replicação Viral/fisiologia , Animais , Humanos , Macaca
14.
Lancet HIV ; 7(7): e504-e513, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32621876

RESUMO

High rates of cognitive disorders in antiretroviral-treated people living with HIV have been described worldwide. The exact prevalence of such cognitive disorders is determined by the definitions used, and the presence of these cognitive disorders significantly impacts the overall wellbeing of people with HIV. With the cohort of people with HIV becoming increasingly older, and having high rates of comorbidities and concomitant medication use, rates of cognitive disorders are likely to increase. Conversely, interventions are being sought to reduce the size of the latent HIV reservoir. If successful, such interventions are likely to also reduce the HIV reservoir in the brain compartment, which could result in improvements in cognitive function and reduced rates of impairment.


Assuntos
Antirretrovirais/uso terapêutico , Transtornos Cognitivos/epidemiologia , Infecções por HIV/complicações , Encéfalo/virologia , Cognição , Transtornos Cognitivos/complicações , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/prevenção & controle , Estudos de Coortes , Comorbidade , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Prevalência , Latência Viral
15.
Nat Commun ; 11(1): 3345, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620802

RESUMO

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved RNA decay mechanism that has emerged as a potent cell-intrinsic restriction mechanism of retroviruses and positive-strand RNA viruses. However, whether NMD is capable of restricting DNA viruses is not known. The DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that NMD restricts KSHV lytic reactivation. Leveraging high-throughput transcriptomics we identify NMD targets transcriptome-wide in PEL cells and identify host and viral RNAs as substrates. Moreover, we identified an NMD-regulated link between activation of the unfolded protein response and transcriptional activation of the main KSHV transcription factor RTA, itself an NMD target. Collectively, our study describes an intricate relationship between cellular targets of an RNA quality control pathway and KSHV lytic gene expression, and demonstrates that NMD can function as a cell intrinsic restriction mechanism acting upon DNA viruses.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Viral/metabolismo , Ativação Viral/genética , Linhagem Celular Tumoral , Células HEK293 , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , RNA Mensageiro/metabolismo , RNA-Seq , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Resposta a Proteínas não Dobradas/genética , Latência Viral/genética
16.
Proc Natl Acad Sci U S A ; 117(31): 18692-18700, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690683

RESUMO

A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1+ adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/106 CD4+ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/106 CD4+ T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.


Assuntos
DNA Viral/sangue , Infecções por HIV , Provírus/genética , Latência Viral/genética , Adulto , Feminino , Infecções por HIV/sangue , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos
17.
Proc Natl Acad Sci U S A ; 117(31): 18764-18770, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694203

RESUMO

Human progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation along the myeloid lineage triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is reexpression of viral major immediate early (MIE) genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34+ HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation.


Assuntos
Citomegalovirus , Fatores de Transcrição Forkhead/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Virais/metabolismo , Citomegalovirus/genética , Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Genes Precoces/genética , Células HeLa , Humanos , Latência Viral
18.
PLoS Pathog ; 16(7): e1008795, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716975

RESUMO

HSV-1 causes 50% of first-time genital herpes infections in resource-rich countries and affects 190 million people worldwide. A prophylactic herpes vaccine is needed to protect against genital infections by both HSV-1 and HSV-2. Previously our laboratory developed a trivalent vaccine that targets glycoproteins C, D, and E present on the HSV-2 virion. We reported that this vaccine protects animals from genital disease and recurrent virus shedding following lethal HSV-2 challenge. Importantly the vaccine also generates cross-reactive antibodies that neutralize HSV-1, suggesting it may provide protection against HSV-1 infection. Here we compared the efficacy of this vaccine delivered as protein or nucleoside-modified mRNA immunogens against vaginal HSV-1 infection in mice. Both the protein and mRNA vaccines protected mice from HSV-1 disease; however, the mRNA vaccine provided better protection as measured by lower vaginal virus titers post-infection. In a second experiment, we compared protection provided by the mRNA vaccine against intravaginal challenge with HSV-1 or HSV-2. Vaccinated mice were totally protected against death, genital disease and infection of dorsal root ganglia caused by both viruses, but somewhat better protected against vaginal titers after HSV-2 infection. Overall, in the two experiments, the mRNA vaccine prevented death and genital disease in 54/54 (100%) mice infected with HSV-1 and 20/20 (100%) with HSV-2, and prevented HSV DNA from reaching the dorsal root ganglia, the site of virus latency, in 29/30 (97%) mice infected with HSV-1 and 10/10 (100%) with HSV-2. We consider the HSV-2 trivalent mRNA vaccine to be a promising candidate for clinical trials for prevention of both HSV-1 and HSV-2 genital herpes.


Assuntos
Proteção Cruzada/imunologia , Herpes Genital , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Vacinas contra Herpesvirus/imunologia , Latência Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Feminino , Herpes Genital/virologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro , Proteínas do Envelope Viral/imunologia
19.
Proc Natl Acad Sci U S A ; 117(27): 15763-15771, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571938

RESUMO

HIV-1 latency is a major barrier to cure. Identification of small molecules that destabilize latency and allow immune clearance of infected cells could lead to treatment-free remission. In vitro models of HIV-1 latency involving cell lines or primary cells have been developed for characterization of HIV-1 latency and high-throughput screening for latency-reversing agents (LRAs). We have shown that the majority of LRAs identified to date are relatively ineffective in cells from infected individuals despite activity in model systems. We show here that, for diverse LRAs, latency reversal observed in model systems involves a heat shock factor 1 (HSF1)-mediated stress pathway. Small-molecule inhibition of HSF1 attenuated HIV-1 latency reversal by histone deactylase inhibitors, protein kinase C agonists, and proteasome inhibitors without interfering with the known mechanism of action of these LRAs. However, latency reversal by second mitochondria-derived activator of caspase (SMAC) mimetics was not affected by inhibition of HSF1. In cells from infected individuals, inhibition of HSF1 attenuated latency reversal by phorbol ester+ionomycin but not by anti-CD3+anti-CD28. HSF1 promotes elongation of HIV-1 RNA by recruiting P-TEFb to the HIV-1 long terminal repeat (LTR), and we show that inhibition of HSF1 attenuates the formation of elongated HIV-1 transcripts. We demonstrate that in vitro models of latency have higher levels of the P-TEFb subunit cyclin T1 than primary cells, which may explain why many LRAs are functional in model systems but relatively ineffective in primary cells. Together, these studies provide insights into why particular LRA combinations are effective in reversing latency in cells from infected individuals.


Assuntos
Infecções por HIV/genética , HIV-1/genética , Fatores de Transcrição de Choque Térmico/genética , Latência Viral/genética , Fármacos Anti-HIV/farmacologia , Proteínas Reguladoras de Apoptose/genética , Ciclina T/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Humanos , Proteínas Mitocondriais/genética , Fator B de Elongação Transcricional Positiva/genética , Proteína Quinase C/genética , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Sequências Repetidas Terminais/genética , Ativação Viral/genética
20.
PLoS Pathog ; 16(6): e1008590, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542010

RESUMO

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.


Assuntos
Infecções por Vírus Epstein-Barr , Antígenos Nucleares do Vírus Epstein-Barr/genética , Deleção de Genes , Herpesvirus Humano 4/fisiologia , Doença de Hodgkin , Linfoma Difuso de Grandes Células B , Proteínas Virais/genética , Latência Viral , Animais , Linhagem Celular , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/patogenicidade , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/virologia , Camundongos , Proteínas Virais/metabolismo
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