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1.
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
2.
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
3.
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
4.
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
5.
Nat Commun ; 11(1): 877, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054837

RESUMO

Epstein-Barr virus (EBV) genomes persist in latently infected cells as extrachromosomal episomes that attach to host chromosomes through the tethering functions of EBNA1, a viral encoded sequence-specific DNA binding protein. Here we employ circular chromosome conformation capture (4C) analysis to identify genome-wide associations between EBV episomes and host chromosomes. We find that EBV episomes in Burkitt's lymphoma cells preferentially associate with cellular genomic sites containing EBNA1 binding sites enriched with B-cell factors EBF1 and RBP-jK, the repressive histone mark H3K9me3, and AT-rich flanking sequence. These attachment sites correspond to transcriptionally silenced genes with GO enrichment for neuronal function and protein kinase A pathways. Depletion of EBNA1 leads to a transcriptional de-repression of silenced genes and reduction in H3K9me3. EBV attachment sites in lymphoblastoid cells with different latency type show different correlations, suggesting that host chromosome attachment sites are functionally linked to latency type gene expression programs.


Assuntos
Sítios de Ligação Microbiológicos/genética , Sítios de Ligação Microbiológicos/fisiologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/virologia , Epigênese Genética , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Herpesvirus Humano 4/patogenicidade , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Modelos Biológicos , Plasmídeos/genética , Latência Viral/genética , Latência Viral/fisiologia
6.
Virology ; 542: 40-53, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32056667

RESUMO

Latent HIV reservoir is a major barrier to absolute HIV cure. Studies on latency reversal agents (LRA) have by far focused mainly on CD4+ T-lymphocytes, while myeloid reservoirs remain under-represented despite their persistence and key contribution to HIV pathogenesis. cAMP has been shown to increase HIV-1 transcription in latently-infected monocytes/macrophages. In this communication, we explored the potential of commercially available pharmacological drugs and phosphodiesterase inhibitors to reactivate HIV in latently-infected monocytic cell-line, U1. We showed that increased levels of intracellular cAMP reverse HIV latency in vitro, which is specific to cells of the myeloid lineage. High throughput RNA-seq analysis revealed that cAMP modulates transcriptional profile of latently HIV-infected cells and provides favourable cellular environment for HIV to produce viral proteins. This reactivation of latent HIV was inhibited by Mithramycin A, a selective Sp1 inhibitor, indicating that the reversal of HIV latency in monocytes is driven by transcription factor Sp1.


Assuntos
HIV-1/genética , HIV-1/fisiologia , Monócitos/metabolismo , Monócitos/virologia , Fator de Transcrição Sp1/metabolismo , Latência Viral/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genoma Viral , HIV-1/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Células Jurkat , Modelos Biológicos , Monócitos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Células U937 , Ativação Viral/efeitos dos fármacos , Ativação Viral/genética , Ativação Viral/fisiologia , Latência Viral/efeitos dos fármacos , Latência Viral/genética
7.
Arch Virol ; 165(2): 321-330, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31828511

RESUMO

Persistence of human immunodeficiency virus 1 (HIV-1) latency and residual immune activation remain major barriers to treatment in patients receiving highly active antiretroviral therapy (HAART). In the present study, we investigated the molecular mechanisms of persistent HIV infection and residual immune activation in HAART-treated patients. We showed that the expression level of B-cell CLL/lymphoma 11B (BCL11B) was significantly increased in CD4+T cells from HIV-infected patients undergoing HAART, and this was accompanied by increased expression of BCL11B-associated chromatin modifiers and inflammatory factors in comparison to healthy controls and untreated patients with HIV. In vitro assays showed that BCL11B significantly inhibited HIV-1 long terminal repeat (LTR)-mediated transcription. Knockdown of BCL11B resulted in the activation of HIV latent cells, and dissociation of BCL11B and its related chromatin remodeling factors from the HIV LTR. Our findings suggested that increased expression of BCL11B and its related chromatin modifiers contribute to HIV-1 transcriptional silencing, and alteration of BCL11B levels might lead to abnormal transcription and inflammation.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Infecções por HIV/genética , HIV-1/genética , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transcrição Genética/imunologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Adulto , Terapia Antirretroviral de Alta Atividade/métodos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Cromatina/genética , Cromatina/virologia , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/imunologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , Repetição Terminal Longa de HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Masculino , Transcrição Genética/efeitos dos fármacos , Latência Viral/genética , Latência Viral/imunologia
8.
Proc Natl Acad Sci U S A ; 117(1): 635-640, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871148

RESUMO

Abortive viral infections are usually studied in populations of susceptible but nonpermissive cells. Single-cell studies of viral infections have demonstrated that even in susceptible and permissive cell populations, abortive infections can be detected in subpopulations of the infected cells. We have previously identified abortive infections in HeLa cells infected with herpes simplex virus 1 (HSV-1) at high multiplicity of infection (MOI). Here, we tested 4 additional human-derived nonneuronal cell lines (cancerous or immortalized) and found significant subpopulations that remain abortive. To characterize these abortive cells, we recovered cell populations that survived infection with HSV-1 at high MOI. The surviving cells retained proliferative potential and the ability to be reinfected. These recovered cell populations maintained the viral genomes in a quiescent state for at least 5 wk postinfection. Our results indicate that these viral genomes are maintained inside the nucleus, bound to cellular histones and occasionally reactivated to produce new progeny viruses. We conclude that abortive HSV-1 infection is a common feature during infection of nonneuronal cells and results in a latency-like state in the infected cells. Our findings question the longstanding paradigm that alphaherpesviruses can establish spontaneous latency only in neuronal cells and emphasize the stochastic nature of lytic versus latency decision of HSV-1 in nonneuronal cells.


Assuntos
Genoma Viral , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Ativação Viral/genética , Latência Viral/genética , Animais , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Células HeLa , Herpesvirus Humano 1/patogenicidade , Humanos , Análise de Célula Única , Células Vero
9.
Nucleic Acids Res ; 48(5): 2643-2660, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31875221

RESUMO

Tat protein is essential to fully activate HIV transcription and processing of viral mRNA, and therefore determines virus expression in productive replication and the establishment and maintenance of latent infection. Here, we used thermodynamic and structure analyses to define a highly conserved sequence-structure in tat mRNA that functions as Tat IRES modulator of tat mRNA (TIM-TAM). By impeding cap-dependent ribosome progression during authentic spliced tat mRNA translation, TIM-TAM stable structure impacts on timing and level of Tat protein hence controlling HIV production and infectivity along with promoting latency. TIM-TAM also adopts a conformation that mediates Tat internal ribosome entry site (IRES)-dependent translation during the early phases of infection before provirus integration. Our results document the critical role of TIM-TAM in Tat expression to facilitate virus reactivation from latency, with implications for HIV treatment and drug development.


Assuntos
Sequência Conservada , Infecções por HIV/virologia , HIV-1/genética , Sítios Internos de Entrada Ribossomal/genética , RNA Viral/química , RNA Viral/genética , Latência Viral/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Bases , Sequência Conservada/genética , Células HeLa , Humanos , Modelos Biológicos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Provírus/genética , RNA Mensageiro/metabolismo , Ativação Viral
10.
Cell Host Microbe ; 27(1): 104-114.e4, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31866424

RESUMO

Infection with human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality following hematopoietic stem cell transplant (HSCT) because of various hematologic problems, including myelosuppression. Here, we demonstrate that latently expressed HCMV miR-US5-2 downregulates the transcriptional repressor NGFI-A binding protein (NAB1) to induce myelosuppression of uninfected CD34+ hematopoietic progenitor cells (HPCs) through an increase in TGF-ß production. Infection of HPCs with an HCMVΔmiR-US5-2 mutant resulted in decreased TGF-ß expression and restoration of myelopoiesis. In contrast, we show that infected HPCs are refractory to TGF-ß signaling as another HCMV miRNA, miR-UL22A, downregulates SMAD3, which is required for maintenance of latency. Our data suggest that latently expressed viral miRNAs manipulate stem cell homeostasis by inducing secretion of TGF-ß while protecting infected HPCs from TGF-ß-mediated effects on viral latency and reactivation. These observations provide a mechanism through which HCMV induces global myelosuppression following HSCT while maintaining lifelong infection in myeloid lineage cells.


Assuntos
Citomegalovirus , Células-Tronco Hematopoéticas/virologia , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Latência Viral , Antígenos CD34/metabolismo , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/metabolismo , Regulação para Baixo , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Células Mieloides/metabolismo , Células Mieloides/virologia , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Ativação Viral , Latência Viral/genética , Latência Viral/fisiologia
11.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31852788

RESUMO

High rates of wild-type (WT) herpes simplex virus 1 (HSV-1) latency reactivation depend on the anti-apoptotic activities of latency-associated transcript (LAT). Replacing LAT with the baculovirus inhibitor of apoptosis protein (cpIAP) or cellular FLIP (FLICE-like inhibitory protein) gene restored the WT latency reactivation phenotype to that of a LAT-minus [LAT(-)] virus, while similar recombinant viruses expressing interleukin-4 (IL-4) or interferon gamma (IFN-γ) did not. However, HSV-1 recombinant virus expressing cpIAP did not restore all LAT functions. Recently, we reported that a similar recombinant virus expressing CD80 in place of LAT had higher latency reactivation than a LAT-null virus. The present study was designed to determine if this CD80-expressing recombinant virus can restore all LAT functions as observed with WT virus. Our results suggest that overexpression of CD80 fully rescues LAT function in latency reactivation, apoptosis, and immune exhaustion, suggesting that LAT and CD80 have multiple overlapping functions.IMPORTANCE Recurring ocular infections caused by HSV-1 can cause corneal scarring and blindness. A major function of the HSV-1 latency-associated transcript (LAT) is to establish high levels of latency and reactivation, thus contributing to the development of eye disease. Here, we show that the host CD80 T cell costimulatory molecule functions similarly to LAT and can restore the ability of LAT to establish latency, reactivation, and immune exhaustion as well as induce the expression of caspase 3, caspase 8, caspase 9, and Bcl2. Our results suggest that, in contrast to several other previously tested genes, CD80-expressing virus can completely compensate for all known and tested LAT functions.


Assuntos
Apoptose/imunologia , Antígeno B7-1/imunologia , Herpesvirus Humano 1/fisiologia , MicroRNAs/imunologia , RNA Viral/imunologia , Ativação Viral/imunologia , Latência Viral/imunologia , Animais , Apoptose/genética , Antígeno B7-1/genética , Camundongos , MicroRNAs/genética , RNA Viral/genética , Ativação Viral/genética , Latência Viral/genética
12.
mBio ; 10(6)2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796538

RESUMO

Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected cell to optimize latent carriage and reactivation. This is achieved, in part, through the expression of viral genes, including the G-protein-coupled receptor US28. Here, we use an unbiased proteomic screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes are downregulated by US28. We validate that major histocompatibility complex (MHC) class II and two pyrin and HIN domain (PYHIN) proteins, myeloid cell nuclear differentiation antigen (MNDA) and IFI16, are downregulated during experimental latency in primary human CD14+ monocytes. We find that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner but only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early gene expression during latency via NF-κB, a function which explains why downregulation of IFI16 during latency is advantageous for the virus.IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50 to 100% of humans worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals but is a serious cause of mortality and morbidity in the immunocompromised and neonates. In particular, reactivation of HCMV in the transplant setting is a major cause of transplant failure and related disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide insights into potential ways to target the latent viral reservoir in at-risk patient populations.


Assuntos
Infecções por Citomegalovirus/genética , Citomegalovirus/imunologia , Interferons/genética , Latência Viral/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/imunologia , Células HEK293 , Humanos , Interferons/imunologia , Monócitos/imunologia , Monócitos/virologia , Células Mieloides/imunologia , Células Mieloides/virologia , NF-kappa B/genética , NF-kappa B/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Proteômica/métodos , Receptores Acoplados a Proteínas-G/imunologia , Células THP-1 , Proteínas Virais/genética , Proteínas Virais/imunologia , Ativação Viral/genética , Ativação Viral/imunologia , Latência Viral/imunologia
13.
Proc Natl Acad Sci U S A ; 116(51): 25392-25394, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31796588

RESUMO

The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4) establish life-long latency in circulating B cells. The precise determinants that mediate in vivo gammaherpesvirus latency and tumorigenesis remain unclear. The EBV-encoded RNAs (EBERs) are among the first noncoding RNAs ever identified and have been the subject of decades of studies; however, their biological roles during in vivo infection remain unknown. Herein, we use a series of refined virus mutants to define the active isoform of MHV68 noncoding RNA TMER4 and demonstrate that EBV EBER1 functionally conserves this activity in vivo to promote egress of infected B cells from lymph nodes into peripheral circulation.


Assuntos
Gammaherpesvirinae/genética , RNA não Traduzido , RNA Viral , Liberação de Vírus/genética , Animais , Células Cultivadas , Infecções por Herpesviridae/virologia , Camundongos , Conformação de Ácido Nucleico , RNA não Traduzido/química , RNA não Traduzido/genética , RNA não Traduzido/fisiologia , RNA Viral/química , RNA Viral/genética , RNA Viral/fisiologia , Baço/citologia , Baço/virologia , Latência Viral/genética
14.
Retrovirology ; 16(1): 42, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852501

RESUMO

The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus's most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly spliced Tax-orf II mRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.


Assuntos
Regulação Viral da Expressão Gênica , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Proteínas dos Retroviridae/genética , Latência Viral/genética , Animais , Linhagem Celular , Expressão Gênica , Genótipo , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Macaca/virologia , RNA Viral/genética , Proteínas dos Retroviridae/imunologia
15.
PLoS Pathog ; 15(11): e1008164, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738810

RESUMO

The human T cell leukemia virus HTLV-1 establishes a persistent infection in vivo in which the viral sense-strand transcription is usually silent at a given time in each cell. However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1+ individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity was similar in polyclonal HTLV-1+ PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1+ clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an average rate of ~0.1 molecules per hour per HTLV-1+ cell; however, between 20% and 70% of HTLV-1-infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivation is highly heterogeneous and occurs in short, self-terminating bursts.


Assuntos
Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Leucócitos Mononucleares/virologia , Análise de Célula Única/métodos , Proteínas Virais/genética , Ativação Viral/genética , Latência Viral/genética , Células Cultivadas , Regulação Viral da Expressão Gênica , Infecções por HTLV-I/genética , Humanos , Hibridização in Situ Fluorescente , Cinética , Processos Estocásticos , Replicação Viral
16.
PLoS One ; 14(11): e0224879, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31710657

RESUMO

The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naïve T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection.


Assuntos
HIV-1/genética , RNA Longo não Codificante/genética , Latência Viral/genética , Depsipeptídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Humanos , Memória Imunológica , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Linfócitos T/imunologia , Latência Viral/efeitos dos fármacos , Vorinostat/farmacologia
17.
PLoS Pathog ; 15(10): e1007838, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31671162

RESUMO

Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA. To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo. Using co-infection experiments and recombinant viruses, we also show that KSHV's ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.


Assuntos
Herpesvirus Humano 8/genética , Proteínas do Grupo Polycomb/metabolismo , Rhadinovirus/genética , Latência Viral/genética , Animais , Linhagem Celular Transformada , Ilhas de CpG/genética , Epigenoma/genética , Feminino , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Código das Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C
18.
PLoS Pathog ; 15(9): e1008025, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31479497

RESUMO

Kaposi's sarcoma associated herpesvirus (KSHV), like all herpesviruses maintains lifelong persistence with its host genome in latently infected cells with only a small fraction of cells showing signatures of productive lytic replication. Modulation of cellular signaling pathways by KSHV-encoded latent antigens, and microRNAs, as well as some level of spontaneous reactivation are important requirements for establishment of viral-associated diseases. Hypoxia, a prominent characteristic of the microenvironment of cancers, can exert specific effects on cell cycle control, and DNA replication through HIF1α-dependent pathways. Furthermore, hypoxia can induce lytic replication of KSHV. The mechanism by which KSHV-encoded RNAs and antigens regulate cellular and viral replication in the hypoxic microenvironment has yet to be fully elucidated. We investigated replication-associated events in the isogenic background of KSHV positive and negative cells grown under normoxic or hypoxic conditions and discovered an indispensable role of KSHV for sustained cellular and viral replication, through protection of critical components of the replication machinery from degradation at different stages of the process. These include proteins involved in origin recognition, pre-initiation, initiation and elongation of replicating genomes. Our results demonstrate that KSHV-encoded LANA inhibits hypoxia-mediated degradation of these proteins to sustain continued replication of both host and KSHV DNA. The present study provides a new dimension to our understanding of the role of KSHV in survival and growth of viral infected cells growing under hypoxic conditions and suggests potential new strategies for targeted treatment of KSHV-associated cancer.


Assuntos
Antígenos Virais/metabolismo , Respiração Celular/fisiologia , Herpesvirus Humano 8/metabolismo , Proteínas Nucleares/metabolismo , Antígenos Virais/genética , Antígenos Virais/imunologia , Linhagem Celular Tumoral , Herpesvirus Humano 8/patogenicidade , Humanos , Hipóxia/metabolismo , Proteínas Nucleares/imunologia , Sarcoma de Kaposi/virologia , Microambiente Tumoral , Latência Viral/genética , Replicação Viral/genética
19.
Virus Genes ; 55(6): 779-785, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31552622

RESUMO

Epstein-Barr virus (EBV) is a widely prevalent pathogen currently infecting over 90% of the human population and is associated with various lymphomas and carcinomas. Lytic replication of EBV is regulated by the expression of the immediate-early genes BZLF1 and BRLF1. In B lymphocytes, BZLF1 transcripts have been shown to be processed to a fully spliced form, as well as zDelta, a spliced variant containing only the first and third exons. While splice variants have been reported in nasopharyngeal carcinoma biopsies, alternative splicing of BZLF1 in EBV-positive epithelial cell lines has not yet been characterized. In this study, we identified the consistent expression of three distinct BZLF1 transcripts in the EBV-positive epithelial cell lines D98/HR1, AGS-BDneo, and AGS-BX1. These BZLF1 transcripts consisted of not only the normally spliced variant but also a completely unspliced and a spliced variant containing exons one and three only. In contrast, we detected only the normally spliced version of the BZLF1 transcript in B-cell lines (B95-8, IM-9, Raji and Daudi). Previous work has also demonstrated that inhibition of the mTOR pathway, via rapamycin, altered total levels of BZLF1 transcripts. We examined the production of specific transcript variants under rapamycin treatment and found that rapamycin alters the production of transcripts in a cell-type, as well as transcripts in variant-type, manners. The expression of these transcript variants may play a role in modulating the replication cycle of EBV within epithelial cells.


Assuntos
Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Transativadores/genética , Linfócitos B/virologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/virologia , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/patogenicidade , Humanos , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Ativação Viral/genética , Latência Viral/genética
20.
EBioMedicine ; 45: 624-629, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31227439

RESUMO

Latent HIV reservoir is the main obstacle that prevents a cure for HIV-1 (HIV). While antiretroviral therapy is effective in controlling viral replication, it cannot eliminate latent HIV reservoirs in patients. Several strategies have been proposed to combat HIV latency, including bone marrow transplantation to replace blood cells with CCR5-mutated stem cells, gene editing to disrupt the HIV genome, and "Shock and Kill" to reactivate latent HIV followed by an immune clearance. However, high risks and limitations to scale-up in clinics, off-target effects in human genomes or failure to reduce reservoir sizes in patients hampered our current efforts to achieve an HIV cure. This necessitates alternative strategies to control the latent HIV reservoirs. This review will discuss an emerging strategy aimed to deeply silence HIV reservoirs, the development of this concept, its potential and caveats for HIV remission/cure, and prospective directions for silencing the latent HIV, thereby preventing viruses from rebound.


Assuntos
Reservatórios de Doenças/virologia , Infecções por HIV/genética , HIV-1/genética , Latência Viral/genética , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Receptores CCR5/genética , Ativação Viral/genética , Replicação Viral/genética
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