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1.
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
2.
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
3.
PLoS Pathog ; 15(12): e1008192, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31809522

RESUMO

The hypoxia-inducible factor 1 alpha (HIF1α) protein and the hypoxic microenvironment are critical for infection and pathogenesis by the oncogenic gammaherpesviruses (γHV), Kaposi sarcoma herpes virus (KSHV) and Epstein-Barr virus (EBV). However, understanding the role of HIF1α during the virus life cycle and its biological relevance in the context of host has been challenging due to the lack of animal models for human γHV. To study the role of HIF1α, we employed the murine gammaherpesvirus 68 (MHV68), a rodent pathogen that readily infects laboratory mice. We show that MHV68 infection induces HIF1α protein and HIF1α-responsive gene expression in permissive cells. siRNA silencing or drug-inhibition of HIF1α reduce virus production due to a global downregulation of viral gene expression. Most notable was the marked decrease in many viral genes bearing hypoxia-responsive elements (HREs) such as the viral G-Protein Coupled Receptor (vGPCR), which is known to activate HIF1α transcriptional activity during KSHV infection. We found that the promoter of MHV68 ORF74 is responsive to HIF1α and MHV-68 RTA. Moreover, Intranasal infection of HIF1αLoxP/LoxP mice with MHV68 expressing Cre- recombinase impaired virus expansion during early acute infection and affected lytic reactivation in the splenocytes explanted from mice. Low oxygen concentrations accelerated lytic reactivation and enhanced virus production in MHV68 infected splenocytes. Thus, we conclude that HIF1α plays a critical role in promoting virus replication and reactivation from latency by impacting viral gene expression. Our results highlight the importance of the mutual interactions of the oxygen-sensing machinery and gammaherpesviruses in viral replication and pathogenesis.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Infecções por Herpesviridae/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Animais , Camundongos , Rhadinovirus/metabolismo
4.
PLoS Pathog ; 15(12): e1008156, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790497

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Current treatment options for KSHV infection and virus associated diseases are sometimes ineffective, therefore, more effectively antiviral agents are urgently needed. As a herpesvirus, lytic replication is critical for KSHV pathogenesis and oncogenesis. In this study, we have established a high-throughput screening assay by using an inducible KSHV+ cell-line, iSLK.219. After screening a compound library that consisted of 1280 Food and Drug Administration (FDA)-approved drugs, 15 hit compounds that effectively inhibited KSHV virion production were identified, most of which have never been reported with anti-KSHV activities. Interestingly, 3 of these drugs target histamine receptors or signaling. Our data further confirmed that antagonists targeting different histamine receptors (HxRs) displayed excellent inhibitory effects on KSHV lytic replication from induced iSLK.219 or BCBL-1 cells. In contrast, histamine and specific agonists of HxRs promoted viral lytic replication from induced iSLK.219 or KSHV-infected primary cells. Mechanistic studies indicated that downstream MAPK and PI3K/Akt signaling pathways were required for histamine/receptors mediated promotion of KSHV lytic replication. Direct knockdown of HxRs in iSLK.219 cells effectively blocked viral lytic gene expression during induction. Using samples from a cohort of HIV+ patients, we found that the KSHV+ group has much higher levels of histamine in their plasma and saliva than the KSHV- group. Taken together, our data have identified new anti-KSHV agents and provided novel insights into the molecular bases of host factors that contribute to lytic replication and reactivation of this oncogenic herpesvirus.


Assuntos
Antivirais/farmacologia , Herpesvirus Humano 8/efeitos dos fármacos , Histamina/metabolismo , Sarcoma de Kaposi/virologia , Ativação Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Herpesvirus Humano 8/fisiologia , Ensaios de Triagem em Larga Escala , Humanos , Receptores Histamínicos/metabolismo , Transdução de Sinais/fisiologia , Ativação Viral/fisiologia , Latência Viral/efeitos dos fármacos , Latência Viral/fisiologia
5.
PLoS Pathog ; 15(12): e1008185, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790507

RESUMO

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.


Assuntos
Herpesvirus Humano 8/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Ativação Viral/fisiologia , Latência Viral/fisiologia , Replicação Viral/fisiologia , Linhagem Celular , Estresse do Retículo Endoplasmático/fisiologia , Infecções por Herpesviridae/virologia , Humanos
6.
Invest Ophthalmol Vis Sci ; 60(10): 3398-3406, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31387116

RESUMO

Purpose: We previously have reported that ICP22, an immediate early gene of herpes simplex virus type 1 (HSV-1), binds to the CD80 promoter to suppress CD80 expression in antigen-presenting cells, leading to reduced T-cell function and protection. In contrast, overexpression of CD80 exacerbates corneal scarring (CS) in ocularly infected mice. In this study we tested the hypothesis that the absence of ICP22 could increase disease severity. Methods: To test our hypothesis, BALB/c mice were ocularly infected after corneal scarification with a recombinant HSV-1 lacking the ICP22 gene with its parental wild-type (WT) virus (KOS) as a control. Virus replication in the eye, CS, angiogenesis, latency, and reactivation between ICP22 null virus and WT KOS were determined. In addition, expression of IL-2, IL-4, IFN-γ, IFN-α, granzyme A, granzyme B, and perforin by CD4 and CD8 T cells in corneas of infected mice on days 3, 5, 7, 10, 14, 21, and 28 postinfection were determined by flow cytometry. Results: We found similar levels of eye disease and angiogenesis in mice following corneal scarification and ocular infection with the ICP22 null virus or parental WT virus despite reduced virus replication in the eye and reduced latency and reactivation in mice ocularly infected with ICP22 null virus. The similar level of eye disease in ICP22 null virus- and WT virus-infected mice correlated with expression of various proinflammatory cytokines that infiltrated the eye after HSV-1 infection. Conclusions: Our study identified a critical role for ICP22 in HSV-1 pathogenicity and suggests that HSV-1-associated CS is more dependent on host immune responses to infection than to virus replication in the eye. Thus, HSV-1 as means of survival uses ICP22 as a mechanism of immune escape that protects the host from increased pathology.


Assuntos
Substância Própria/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Ceratite Herpética/imunologia , Latência Viral/fisiologia , Animais , Antígeno B7-1/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Imediatamente Precoces/deficiência , Ceratite Herpética/patologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Lágrimas/virologia , Replicação Viral/fisiologia
7.
Surg Pathol Clin ; 12(3): 745-770, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31352986

RESUMO

Although about 90% of the world's population is infected by EBV only a small subset of the related infections result in neoplastic transformation. EBV is a versatile oncogenic agent involved in a multitude of hematopoietic, epithelial, and mesenchymal neoplasms, but the precise role of EBV in the pathogenesis of many of the associated lymphoid/histiocytic proliferations remains hypothetical or not completely understood. Additional studies and use of evolving technologies such as high-throughput next-generation sequencing may help address this knowledge gap and may lead to enhanced diagnostic assessment and the development of potential therapeutic interventions.


Assuntos
Infecções por Vírus Epstein-Barr/classificação , Transtornos Linfoproliferativos/classificação , Animais , Doença Crônica , Culicidae , Diagnóstico Diferencial , Humanos , Hidroa Vaciniforme/diagnóstico , Imunossupressores/efeitos adversos , Mononucleose Infecciosa/diagnóstico , Mordeduras e Picadas de Insetos/diagnóstico , Linfoma de Células B/classificação , Linfoma de Células B/virologia , Linfoma de Células T/classificação , Linfoma de Células T/virologia , Granulomatose Linfomatoide/diagnóstico , Transtornos Linfoproliferativos/virologia , Neoplasias de Plasmócitos/diagnóstico , Prognóstico , Pseudolinfoma/diagnóstico , Pseudolinfoma/virologia , Latência Viral/fisiologia
8.
PLoS Pathog ; 15(6): e1007884, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206552

RESUMO

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Poliadenilação , Precursores de RNA/metabolismo , Processamento de RNA , RNA Viral/metabolismo , Latência Viral/fisiologia , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Precursores de RNA/genética , RNA Viral/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética
9.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 89-101, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102363

RESUMO

Herpes simplex virus (HSV), including HSV-1 and HSV-2, is an important pathogen that can cause many diseases. Usually these diseases are recurrent and incurable. After lytic infection on the surface of peripheral mucosa, HSV can enter sensory neurons and establish latent infection during which viral replication ceases. Moreover, latent virus can re-enter the replication cycle by reactivation and return to peripheral tissues to start recurrent infection. This ability to escape host immune surveillance during latent infection and to spread during reactivation is a viral survival strategy and the fundamental reason why no drug can completely eradicate the virus at present. Although there are many studies on latency and reactivation of HSV, and much progress has been made, many specific mechanisms of the process remain obscure or even controversial due to the complexity of this process and the limitations of research models. This paper reviews the major results of research on HSV latency and reactivation, and discusses future research directions in this field.


Assuntos
Herpes Simples , Herpesvirus Humano 1 , Ativação Viral , Latência Viral , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Humanos , Ativação Viral/fisiologia , Latência Viral/fisiologia , Replicação Viral
10.
Biochem Pharmacol ; 164: 237-251, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30991051

RESUMO

The persistence of latent human immunodeficiency virus type 1 (HIV-1) reservoirs remains a major hurdle for HIV-1 eradication. The "shock and kill" strategy relies on the drug-mediated reversion of HIV-1 latency and the subsequent death of HIV-producing cells. Unfortunately, none of the agents currently in use possess a sufficient potency to reactivate latent virus or eliminate the latent HIV-1 reservoir in vivo. Here, we demonstrated that a promising specific bromodomain and extraterminal domain inhibitor, CPI-203, could potently reactivate latent HIV-1 in different latently infected cell lines with minimal cytotoxicity by activating the positive transcription elongation factor b signaling pathway. Notably, CPI-203 exhibited synergism in latent HIV-1 reactivation and alleviated the HIV-1-induced "cytokine storm" when used in combination with the protein kinase C (PKC) agonist prostratin. These findings highlight that CPI-203 shows promise as a novel, safe candidate for the design of targeted strategies to "shock and kill" HIV-1 and thus represents a potential functional cure.


Assuntos
Acetamidas/farmacologia , Azepinas/farmacologia , HIV-1/efeitos dos fármacos , Fator B de Elongação Transcricional Positiva/metabolismo , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Acetamidas/química , Adulto , Animais , Azepinas/química , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Feminino , HIV-1/fisiologia , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/fisiologia , Ativação Viral/fisiologia , Latência Viral/fisiologia
11.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728262

RESUMO

Herpes simplex virus (HSV) establishes latency in neurons of the peripheral and central nervous systems (CNS). Evidence is mounting that HSV latency and reactivation in the nervous system has the potential to promote neurodegenerative processes. Understanding how this occurs is an important human health goal. In the mouse model, in vivo viral reactivation in the peripheral nervous system, triggered by hyperthermic stress, has been well characterized with respect to frequency and cell type. However, characterization of in vivo reactivation in the CNS is extremely limited. Further, it remains unclear whether virus reactivated in the peripheral nervous system is transported to the CNS in an infectious form, how often this occurs, and what parameters underlie the efficiency and outcomes of this process. In this study, reactivation was quantified in the trigeminal ganglia (TG) and the brain stem from the same latently infected animal using direct assays of equivalent sensitivity. Reactivation was detected more frequently in the TG than in the brain stem and, in all but one case, the amount of virus recovered was greater in the TG than that detected in the brain stem. Viral protein positive neurons were observed in the TG, but a cellular source for reactivation in the brain stem was not identified, despite serially sectioning and examining the entire tissue (0/6 brain stems). These findings suggest that infectious virus detected in the brain stem is primarily the result of transport of reactivated virus from the TG into the brain stem.IMPORTANCE Latent herpes simplex virus (HSV) DNA has been detected in the central nervous systems (CNS) of humans postmortem, and infection with HSV has been correlated with the development of neurodegenerative diseases. However, whether HSV can directly reactivate in the CNS and/or infectious virus can be transported to the CNS following reactivation in peripheral ganglia has been unclear. In this study, infectious virus was recovered from both the trigeminal ganglia and the brain stem of latently infected mice following a reactivation stimulus, but a higher frequency of reactivation and increased titers of infectious virus were recovered from the trigeminal ganglia. Viral proteins were detected in neurons of the trigeminal ganglia, but a cellular source of infectious virus could not be identified in the brain stem. These results suggest that infectious virus is transported from the ganglia to the CNS following reactivation but do not exclude the potential for direct reactivation in the CNS.


Assuntos
Tronco Encefálico/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Gânglio Trigeminal/metabolismo , Proteínas Virais/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Transporte Biológico Ativo , Tronco Encefálico/patologia , Tronco Encefálico/virologia , Feminino , Herpes Simples/patologia , Masculino , Camundongos , Coelhos , Gânglio Trigeminal/patologia , Gânglio Trigeminal/virologia
12.
J Virol ; 93(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30760571

RESUMO

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.


Assuntos
Epitélio Anterior/metabolismo , Herpesvirus Humano 1 , Ceratite Herpética/metabolismo , Proteínas de Ligação a RNA/metabolismo , Gânglio Trigeminal/metabolismo , Proteínas Virais/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Epitélio Anterior/patologia , Epitélio Anterior/virologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Deleção de Genes , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Humanos , Ceratite Herpética/genética , Ceratite Herpética/patologia , Ceratite Herpética/virologia , Fosforilação , Proteínas de Ligação a RNA/genética , Gânglio Trigeminal/patologia , Gânglio Trigeminal/virologia , Células Vero , Proteínas Virais/genética
13.
Curr HIV/AIDS Rep ; 16(1): 96-104, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30734905

RESUMO

PURPOSE OF REVIEW: In addition to preventive protocols and antiretroviral therapy, HIV-1 eradication has been considered as an additional strategy to help fight the AIDS epidemic. With the support of multiple funding agencies, research groups worldwide have been developing protocols to achieve either a sterilizing or a functional cure for HIV-infection. RECENT FINDINGS: Most of the studies focus on the elimination or suppression of circulating CD4+ T cells, the best characterized HIV-1 latent reservoir. The role of the central nervous system (CNS) as a latent reservoir is still controversial. Although brain macrophages and astrocytes are susceptible to HIV-1 infection, it has not been ascertained whether the CNS carries latent HIV-1 during cART and, if so, whether the virus can be reactivated and spread to other compartments after ART interruption. Here, we examine the implications of HIV-1 eradication strategies on the CNS, regardless of whether it is a true latent reservoir and, if so, whether it is present in all patients.


Assuntos
Encéfalo/virologia , Linfócitos T CD4-Positivos/virologia , Erradicação de Doenças/métodos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Latência Viral/fisiologia , Astrócitos/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Reservatórios de Doenças/virologia , HIV-1/efeitos dos fármacos , Humanos , Macrófagos/virologia , Latência Viral/efeitos dos fármacos
14.
J Vis Exp ; (143)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30735158

RESUMO

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the "shock and kill" approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.


Assuntos
Infecções por HIV/virologia , Processamento de RNA/fisiologia , Latência Viral/fisiologia , Humanos
15.
Virol J ; 16(1): 22, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30786885

RESUMO

BACKGROUND: The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized. METHODS: To better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes. RESULTS: In activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues. CONCLUSIONS: CycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication.


Assuntos
Linfócitos T CD4-Positivos/virologia , Ciclina T/genética , Infecções por HIV/imunologia , Latência Viral/fisiologia , Replicação Viral/fisiologia , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo , Regulação da Expressão Gênica , HIV-1/fisiologia , Interações entre Hospedeiro e Microrganismos , Humanos , Fator B de Elongação Transcricional Positiva/genética , Ativação Transcricional
16.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626677

RESUMO

Curing HIV infection has been thwarted by the persistent reservoir of latently infected CD4+ T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir depletion strategies, we developed a novel preclinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, 2 weeks after PBMC injection, was 1,000-fold higher (mean = 9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean = 6,838 HIV copies/ml) or intravenously (mean = 591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo-reactivated primary latently infected cells. The time to viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, compared to treatment with 10-1074-FcRnull, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated, or 10-1074-FcRnull-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in the plasma of 10-1074-treated mice, whereas two new clusters emerged that were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time to viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral/fisiologia , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Modelos Animais de Doenças , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Camundongos , Filogenia , Baço/imunologia , Baço/virologia , Carga Viral/imunologia , Carga Viral/fisiologia , Viremia/imunologia , Viremia/virologia , Latência Viral/imunologia , Replicação Viral/imunologia
17.
PLoS Pathog ; 15(1): e1007498, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30645648

RESUMO

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.


Assuntos
HIV-1/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Latência Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Soropositividade para HIV , HIV-1/patogenicidade , Humanos , Células Jurkat , Complexo de Endopeptidases do Proteassoma/fisiologia , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Fatores de Elongação da Transcrição , Ativação Viral/efeitos dos fármacos , Latência Viral/fisiologia
18.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602607

RESUMO

Lund human mesencephalic (LUHMES) cells are human embryonic neuronal precursor cells that can be maintained as proliferating cells due to the expression of a tetracycline-regulatable (Tet-off) v-myc transgene. They can be differentiated to postmitotic neurons by the addition of tetracycline, glial cell-derived neurotrophic factor (GDNF), and dibutyryl cAMP. We demonstrate that these cells can be infected with herpes simplex virus 1 (HSV-1) at a multiplicity of infection (MOI) of 3 with the majority of cells surviving. By 6 days postinfection, there is a loss of lytic gene transcription and an increase in the numbers of neurons that express the latency-associated transcripts (LATs). Importantly, the virus can then be reactivated by the addition of a phosphoinositide 3-kinase inhibitor, which has previously been shown to reactivate HSV-1 in rat neuron cultures. While rodent primary culture neuron systems have been described, these are limited by their lack of scalability, as it is difficult to obtain more than 500,000 neurons to employ for a given experiment. Several recent papers have described a human dorsal root ganglion (DRG) neuron culture model and human induced pleuripotent stem cell (iPSC) neuron culture models that are scalable, but they require that the presence of an antiviral suppression be maintained following HSV-1 infection. The human LUHMES cell model of HSV-1 infection described here may be especially useful for studying HSV-1 latency and reactivation on account of its scalability, its amenability to maintenance of latency without the continual use of antiviral inhibitors, and its latent gene expression profile which mirrors many properties observed in vivo, importantly, the heterogeneity of cells expressing the LATs.IMPORTANCE Herpes simplex virus (HSV) is responsible for significant morbidity in humans due to its ability to cause oral and genital lesions, ocular disease, and encephalitis. While antivirals can attenuate the severity and frequency of disease, there is no vaccine or cure. Understanding the molecular details of HSV latency and reactivation is key to the development of new therapies. One of the difficulties in studying HSV latency has been the need to rely on establishment of latent infections in animal models. While rodent primary neuron culture models have shown promise, they yield relatively small numbers of latently infected neurons for biochemical and molecular analyses. Here we present the use of a human central nervous system (CNS)-derived conditionally proliferating cell line that can be differentiated into mature neurons and latently infected with HSV-1. This model shows promise as a scalable tool to study molecular and biochemical aspects of HSV-1 latency and reactivation in human neurons.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Neurônios/virologia , Latência Viral/fisiologia , Linhagem Celular , Gânglios Espinais/virologia , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Transcrição Genética/fisiologia , Ativação Viral/fisiologia , Replicação Viral/fisiologia
19.
Clin Pharmacol Ther ; 105(3): 692-702, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30137649

RESUMO

Disulfiram (DSF) was well tolerated and activated viral transcription (cell-associated unspliced (CA-US) and plasma human immunodeficiency virus (HIV) RNA) in a phase II dose-escalation trial in HIV+ antiretroviral therapy (ART)-suppressed participants. Here, we investigated whether exposure to DSF and its metabolites predicted these changes in HIV transcription. Participants were administered 500 (N = 10), 1,000 (N = 10), or 2,000 (N = 10) mg of DSF for 3 consecutive days. DSF and four metabolites were measured by ultraperformance liquid chromatography-tandem mass spectrometry. Changes in CA-US and plasma HIV RNA were quantified by polymerase chain reaction (PCR) and analyzed in NONMEM. A seven-compartment pharmacokinetic (PK) model demonstrated nonlinear elimination kinetics. The fitted median area under the curve values for 72 hours (AUC0-72 ) were 3,816, 8,386, and 22,331 mg*hour/L, respectively. Higher exposure predicted greater increases in CA-US (maximum effect (Emax ) = 78%, AUC50  = 1,600 µg*hour/L, P = 0.013) but not plasma HIV RNA. These results provide support for further development of DSF as an important drug for future HIV cure strategies.


Assuntos
Dissulfiram/farmacocinética , Infecções por HIV/sangue , HIV-1/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Inibidores de Acetaldeído Desidrogenases/farmacocinética , Inibidores de Acetaldeído Desidrogenases/uso terapêutico , Adulto , Idoso , Dissulfiram/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem/métodos , Transcrição Genética/fisiologia , Latência Viral/fisiologia
20.
Expert Rev Clin Pharmacol ; 12(1): 17-29, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30570410

RESUMO

Introduction: Combined antiretroviral therapy (cART) has transformed an inexorably fatal disease into a chronic pathology, shifting the focus of research from the control of viral replication to the possibility of HIV cure. Areas covered: The present review assesses the principal pharmacological strategies that have been tested for an HIV cure starting from the in vitro proof of concept and the potential rationale of their in vivo applicability. We evaluated the possible pharmacological procedures employed during the early-stage HIV infection and the possibility of cART-free remission. We then analyzed the shock and kill approach from the single compounds in vitro mechanism of action, to the in vivo application of single or combined actions. Finally, we briefly considered the novel immunological branch through the discovery and development of broadly neutralizing antibodies in regard to the current and future in vivo therapeutic strategies aiming to verify the clinical applicability of these compounds. Expert opinion: Despite an incredible effort in HIV research cure, the likelihood of completely eradicating HIV is unreachable within our current knowledge. A better understanding of the mechanism of viral latency and the full characterization of HIV reservoir are crucial for the discovery of new therapeutic targets and novel pharmacological entities.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Animais , Fármacos Anti-HIV/farmacologia , Anticorpos Neutralizantes/imunologia , Descoberta de Drogas/métodos , Quimioterapia Combinada , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Terapia de Alvo Molecular , Latência Viral/fisiologia
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