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1.
J Med Virol ; 96(5): e29684, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38773828

RESUMO

Kaposi's sarcoma (KS) may derive from Kaposi's sarcoma herpesvirus (KSHV)-infected human mesenchymal stem cells (hMSCs) that migrate to sites characterized by inflammation and angiogenesis, promoting the initiation of KS. By analyzing the RNA sequences of KSHV-infected primary hMSCs, we have identified specific cell subpopulations, mechanisms, and conditions involved in the initial stages of KSHV-induced transformation and reprogramming of hMSCs into KS progenitor cells. Under proangiogenic environmental conditions, KSHV can reprogram hMSCs to exhibit gene expression profiles more similar to KS tumors, activating cell cycle progression, cytokine signaling pathways, endothelial differentiation, and upregulating KSHV oncogenes indicating the involvement of KSHV infection in inducing the mesenchymal-to-endothelial (MEndT) transition of hMSCs. This finding underscores the significance of this condition in facilitating KSHV-induced proliferation and reprogramming of hMSCs towards MEndT and closer to KS gene expression profiles, providing further evidence of these cell subpopulations as precursors of KS cells that thrive in a proangiogenic environment.


Assuntos
Herpesvirus Humano 8 , Células-Tronco Mesenquimais , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/virologia , Células-Tronco Mesenquimais/virologia , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Proliferação de Células
2.
Viruses ; 16(5)2024 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-38793630

RESUMO

During viral infection, the innate immune system utilizes a variety of specific intracellular sensors to detect virus-derived nucleic acids and activate a series of cellular signaling cascades that produce type I IFNs and proinflammatory cytokines and chemokines. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus that has been associated with a variety of human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. Infection with KSHV activates various DNA sensors, including cGAS, STING, IFI16, and DExD/H-box helicases. Activation of these DNA sensors induces the innate immune response to antagonize the virus. To counteract this, KSHV has developed countless strategies to evade or inhibit DNA sensing and facilitate its own infection. This review summarizes the major DNA-triggered sensing signaling pathways and details the current knowledge of DNA-sensing mechanisms involved in KSHV infection, as well as how KSHV evades antiviral signaling pathways to successfully establish latent infection and undergo lytic reactivation.


Assuntos
DNA Viral , Herpesvirus Humano 8 , Imunidade Inata , Transdução de Sinais , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Humanos , DNA Viral/metabolismo , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/metabolismo , Sarcoma de Kaposi/virologia , Nucleotidiltransferases/metabolismo , Interações Hospedeiro-Patógeno , Animais , Proteínas de Membrana/metabolismo , Proteínas Nucleares , Fosfoproteínas
3.
J Virol ; 98(6): e0025524, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38752725

RESUMO

Human herpesvirus 8 (HHV-8), associated with Kaposi sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman disease, encodes four interferon regulatory factor homologs, vIRFs 1-4, that interact with and inhibit various mediators of host-cell defense against virus infection. A cellular protein targeted by all the vIRFs is ubiquitin-specific protease 7 (USP7); while replication-modulatory and latently infected PEL-cell pro-viability phenotypes of USP7 targeting have been identified for vIRFs 1-3, the significance of the interaction of vIRF-4 with USP7 has remained undetermined. Here we show, through genetic ablation of the vIRF-4-USP7 interaction in infected cells, that vIRF-4 association with USP7 is necessary for optimal expression of vIRF-4 and normal HHV-8 replication. Findings from experiments on transfected and infected cells identified ubiquitination of vIRF-4 via K48-linkage and USP7-binding-associated suppression of vIRF-4 ubiquitination and, in infected cells, increased vIRF-4 expression. Analysis of IFN-I induction and associated signaling as a function of vIRF-4 and its interaction with USP7 identified a role of each in innate-immune suppression. Finally, activation via K63-polyubiquitination of the innate-immune signaling mediator TRAF3 was found to be suppressed by vIRF-4 in a USP7-binding-associated manner in infected cells, but not in transfected cells, likely via binding-regulated expression of vIRF-4. Together, our data identify the first examples of vIRF ubiquitination and a vIRF substrate of USP7, enhanced expression of vIRF-4 via its interaction with USP7, and TRAF3-inhibitory activity of vIRF-4. The findings address, for the first time, the biological significance of the interaction of vIRF-4 with USP7 and reveal a mechanism of vIRF-4-mediated innate-immune evasion and pro-replication activity via TRAF3 regulation. IMPORTANCE: HHV-8 homologs of cellular interferon regulatory factors (IRFs), involved in host-cell defense against virus infection, interact in an inhibitory fashion with IRFs and other mediators of antiviral innate immunity. These interactions are of demonstrated or hypothesized importance for successful primary, productive (lytic), and latent (persistent) infection by HHV-8. While HHV-8 vIRF-4 is known to interact physically with USP7 deubiquitinase, a key regulator of various cellular proteins, the functional and biological significance of the interaction has not been addressed. The present study identifies the interaction as important for HHV-8 productive replication and, indeed, for vIRF-4 expression and reveals a new function of vIRF-4 via inhibition of the activity of TRAF3, a pivotal mediator of host-cell antiviral activity through activation of cellular IRFs and induction of type-I interferons. These findings identify potential targets for the development of novel anti-HHV-8 agents, such as those able to disrupt vIRF-4-USP7 interaction or vIRF-4-stabilizing USP7 activity.


Assuntos
Herpesvirus Humano 8 , Fatores Reguladores de Interferon , Peptidase 7 Específica de Ubiquitina , Ubiquitinação , Proteínas Virais , Replicação Viral , Humanos , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Células HEK293 , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Ligação Proteica , Interações Hospedeiro-Patógeno
4.
J Virol ; 98(6): e0057624, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38767375

RESUMO

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causal agent of Kaposi sarcoma, a cancer that appears as tumors on the skin or mucosal surfaces, as well as primary effusion lymphoma and KSHV-associated multicentric Castleman disease, which are B-cell lymphoproliferative disorders. Effective prophylactic and therapeutic strategies against KSHV infection and its associated diseases are needed. To develop these strategies, it is crucial to identify and target viral glycoproteins involved in KSHV infection of host cells. Multiple KSHV glycoproteins expressed on the viral envelope are thought to play a pivotal role in viral infection, but the infection mechanisms involving these glycoproteins remain largely unknown. We investigated the role of two KSHV envelope glycoproteins, KSHV complement control protein (KCP) and K8.1, in viral infection in various cell types in vitro and in vivo. Using our newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP, K8.1, or both, we demonstrated the presence of KCP and K8.1 on the surface of both virions and KSHV-infected cells. We showed that KSHV lacking KCP and/or K8.1 remained infectious in KSHV-susceptible cell lines, including epithelial, endothelial, and fibroblast, when compared to wild-type recombinant KSHV. We also provide the first evidence that KSHV lacking K8.1 or both KCP and K8.1 can infect human B cells in vivo in a humanized mouse model. Thus, these results suggest that neither KCP nor K8.1 is required for KSHV infection of various host cell types and that these glycoproteins do not determine KSHV cell tropism. IMPORTANCE: Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human gamma-herpesvirus associated with the endothelial malignancy Kaposi sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. Determining how KSHV glycoproteins such as complement control protein (KCP) and K8.1 contribute to the establishment, persistence, and transmission of viral infection will be key for developing effective anti-viral vaccines and therapies to prevent and treat KSHV infection and KSHV-associated diseases. Using newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP and/or K8.1, we show that KCP and K8.1 can be found on the surface of both virions and KSHV-infected cells. Furthermore, we show that KSHV lacking KCP and/or K8.1 remains infectious to diverse cell types susceptible to KSHV in vitro and to human B cells in vivo in a humanized mouse model, thus providing evidence that these viral glycoproteins are not required for KSHV infection.


Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Proteínas do Envelope Viral , Proteínas Virais , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Humanos , Animais , Camundongos , Proteínas Virais/metabolismo , Proteínas Virais/genética , Sarcoma de Kaposi/virologia , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Linhagem Celular , Hiperplasia do Linfonodo Gigante/virologia , Hiperplasia do Linfonodo Gigante/metabolismo , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/metabolismo , Células HEK293 , Células Endoteliais/virologia
5.
J Virol ; 98(6): e0000524, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38717113

RESUMO

TRIM32 is often aberrantly expressed in many types of cancers. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi's sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers. IMPORTANCE: TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi's sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers.


Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Transativadores , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Replicação Viral , Humanos , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Transativadores/metabolismo , Transativadores/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteólise , Latência Viral , Apoptose , Ativação Viral , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Linfoma de Efusão Primária/virologia , Linfoma de Efusão Primária/metabolismo
6.
PLoS Pathog ; 20(4): e1012141, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38626263

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus etiologically associated with multiple malignancies. Both latency and sporadic lytic reactivation contribute to KSHV-associated malignancies, however, the specific roles of many KSHV lytic gene products in KSHV replication remain elusive. In this study, we report that ablation of ORF55, a late gene encoding a tegument protein, does not impact KSHV lytic reactivation but significantly reduces the production of progeny virions. We found that cysteine 10 and 11 (C10 and C11) of pORF55 are palmitoylated, and the palmytoilation is essential for its Golgi localization and secondary envelope formation. Palmitoylation-defective pORF55 mutants are unstable and undergo proteasomal degradation. Notably, introduction of a putative Golgi localization sequence to these palmitoylation-defective pORF55 mutants restores Golgi localization and fully reinstates KSHV progeny virion production. Together, our study provides new insight into the critical role of pORF55 palmitoylation in KSHV progeny virion production and offers potential therapeutic targets for the treatment of related malignancies.


Assuntos
Complexo de Golgi , Herpesvirus Humano 8 , Lipoilação , Proteínas Virais , Vírion , Replicação Viral , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Humanos , Vírion/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Replicação Viral/fisiologia , Células HEK293
7.
J Med Virol ; 96(3): e29534, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38501356

RESUMO

Human endogenous retrovirus sequences (HERVs) constitute up to 8% of the human genome, yet not all HERVs remain silent passengers within our genomes. Some HERVs, especially the HERV type K (HERV-K), have been found to be frequently transactivated in a variety of inflammatory diseases and human cancers. Np9, a 9-kDa HERV-K encoded protein, has been reported as an oncoprotein and found present in a variety of tumors and transformed cells. In the current study, we for the first time reported that ectopic expression of Np9 protein was able to induce DNA damage response from host cells especially through upregulation of γH2AX. Furthermore, we found that direct knockdown of Np9 by RNAi in Kaposi's Sarcoma-associated herpesvirus (KSHV) infected cells effectively reduced LANA expression, the viral major latent oncoprotein in vitro and in vivo, which may represent a novel strategy against virus-associated malignancies.


Assuntos
Retrovirus Endógenos , Herpesvirus Humano 8 , Neoplasias , Humanos , Retrovirus Endógenos/genética , Herpesvirus Humano 8/fisiologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Reparo do DNA
8.
Cell Rep ; 43(3): 113888, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38416644

RESUMO

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.


Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Estudo de Associação Genômica Ampla , Antígenos Virais/genética , Antígenos Virais/metabolismo , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica , Latência Viral
9.
J Med Virol ; 96(2): e29436, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38380509

RESUMO

Kaposi sarcoma (KS), caused by Herpesvirus-8 (HHV-8; KSHV), shows sporadic, endemic, and epidemic forms. While familial clustering of KS was previously recorded, the molecular basis of hereditary predilection to KS remains largely unknown. We demonstrate through genetic studies that a dominantly inherited missense mutation in BPTF segregates with a phenotype of classical KS in multiple immunocompetent individuals in two families. Using an rKSHV.219-infected CRISPR/cas9-model, we show that BPTFI2012T mutant cells exhibit higher latent-to-lytic ratio, decreased virion production, increased LANA staining, and latent phenotype in viral transcriptomics. RNA-sequencing demonstrated that KSHV infection dysregulated oncogenic-like response and P53 pathways, MAPK cascade, and blood vessel development pathways, consistent with KS. BPTFI2012T also enriched pathways of viral genome regulation and replication, immune response, and chemotaxis, including downregulation of IFI16, SHFL HLAs, TGFB1, and HSPA5, all previously associated with KSHV infection and tumorigenesis. Many of the differentially expressed genes are regulated by Rel-NF-κB, which regulates immune processes, cell survival, and proliferation and is pivotal to oncogenesis. We thus demonstrate BPTF mutation-mediated monogenic hereditary predilection of KSHV virus-induced oncogenesis, and suggest BPTF as a drug target.


Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Carcinogênese , Herpesvirus Humano 8/fisiologia , NF-kappa B/metabolismo , Sarcoma de Kaposi/genética , Latência Viral/genética , Replicação Viral
10.
PLoS Pathog ; 20(1): e1011907, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38232124

RESUMO

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.


Assuntos
Herpesvirus Humano 8 , Proteínas Nucleares , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Latência Viral/genética , Antígenos Virais/genética , Antígenos Virais/metabolismo , Expressão Gênica , Regulação Viral da Expressão Gênica , Replicação Viral
11.
PLoS Pathog ; 20(1): e1011881, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38190392

RESUMO

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKÆ´ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.


Assuntos
Infecções por HIV , Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Células Endoteliais/metabolismo , Infecções por HIV/metabolismo , Isoformas de Proteínas/metabolismo , Microambiente Tumoral
12.
J Virol ; 98(2): e0138623, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38240593

RESUMO

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.


Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Histonas/genética , Histonas/metabolismo , Nucleossomos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Latência Viral/genética , Antígenos Virais/genética , Antígenos Virais/metabolismo , Sequências Repetidas Terminais/genética , Regulação Viral da Expressão Gênica
13.
J Virol ; 98(2): e0156723, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38197631

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family, which can cause human malignancies including Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman's diseases. KSHV typically maintains a persistent latent infection within the host. However, after exposure to intracellular or extracellular stimuli, KSHV lytic replication can be reactivated. The reactivation process of KSHV triggers the innate immune response to limit viral replication. Here, we found that the transcriptional regulator RUNX3 is transcriptionally upregulated by the NF-κB signaling pathway in KSHV-infected SLK cells and B cells during KSHV reactivation. Notably, knockdown of RUNX3 significantly promotes viral lytic replication as well as the gene transcription of KSHV. Consistent with this finding, overexpression of RUNX3 impairs viral lytic replication. Mechanistically, RUNX3 binds to the KSHV genome and limits viral replication through transcriptional repression, which is related to its DNA- and ATP-binding ability. However, KSHV has also evolved corresponding strategies to antagonize this inhibition by using the viral protein RTA to target RUNX3 for ubiquitination and proteasomal degradation. Altogether, our study suggests that RUNX3, a novel host-restriction factor of KSHV that represses the transcription of viral genes, may serve as a potential target to restrict KSHV transmission and disease development.IMPORTANCEThe reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latent infection to lytic replication is important for persistent viral infection and tumorigenicity. However, reactivation is a complex event, and the regulatory mechanisms of this process are not fully elucidated. Our study revealed that the host RUNX3 is upregulated by the NF-κB signaling pathway during KSHV reactivation, which can repress the transcription of KSHV genes. At the late stage of lytic replication, KSHV utilizes a mechanism involving RTA to degrade RUNX3, thus evading host inhibition. This finding helps elucidate the regulatory mechanism of the KSHV life cycle and may provide new clues for the development of therapeutic strategies for KSHV-associated diseases.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core , Herpesvirus Humano 8 , Infecção Latente , Humanos , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 8/fisiologia , NF-kappa B/metabolismo , Ativação Viral , Latência Viral , Replicação Viral , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo
14.
J Virol ; 98(2): e0126823, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38240588

RESUMO

Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.


Assuntos
Antígenos Virais , Herpesvirus Humano 8 , Plasmídeos , Sarcoma de Kaposi , Humanos , Antígenos Virais/metabolismo , DNA , Herpesvirus Humano 8/fisiologia , Ácidos Indolacéticos , Nucleotidiltransferases/genética , Sarcoma de Kaposi/virologia , Latência Viral , Proteínas Nucleares/metabolismo
15.
mBio ; 15(1): e0277423, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38095447

RESUMO

IMPORTANCE: Hypoxia can induce the reactivation of Kaposi sarcoma-associated virus (KSHV), which necessitates the synthesis of critical structural proteins. Despite the unfavorable energetic conditions of hypoxia, KSHV utilizes mechanisms to prevent the degradation of essential cellular machinery required for successful reactivation. Our study provides new insights on strategies employed by KSHV-infected cells to maintain steady-state transcription by overcoming hypoxia-mediated metabolic stress to enable successful reactivation. Our discovery that the interaction of latency-associated nuclear antigen with HIF1α and NEDD4 inhibits its polyubiquitination activity, which blocks the degradation of RNA Pol II during hypoxia, is a significant contribution to our understanding of KSHV biology. This newfound knowledge provides new leads in the development of novel therapies for KSHV-associated diseases.


Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Latência Viral/genética , Antígenos Virais/genética , Hipóxia/metabolismo , Replicação Viral
16.
mBio ; 15(1): e0301123, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38117084

RESUMO

IMPORTANCE: Kaposi's sarcoma (KS) is the most common cancer in HIV-infected patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Hyperinflammation is the hallmark of KS. In this study, we have shown that KSHV mediates hyperinflammation by inducing IL-1α and suppressing IL-1Ra. Mechanistically, KSHV miRNAs and vFLIP induce hyperinflammation by activating the NF-κB pathway. A common anti-inflammatory agent dexamethasone blocks KSHV-induced hyperinflammation and tumorigenesis by activating glucocorticoid receptor signaling to suppress IL-1α and induce IL-1Ra. This work has identified IL-1-mediated inflammation as a potential therapeutic target and dexamethasone as a potential therapeutic agent for KSHV-induced malignancies.


Assuntos
Transformação Celular Neoplásica , Dexametasona , Herpesvirus Humano 8 , Receptores de Glucocorticoides , Sarcoma de Kaposi , Humanos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Herpesvirus Humano 8/fisiologia , Inflamação/virologia , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Receptores de Glucocorticoides/metabolismo , Sarcoma de Kaposi/tratamento farmacológico
17.
Viruses ; 15(12)2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-38140679

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes life-long latent infection and is linked to several human malignancies. Latency-associated nuclear antigen (LANA) is highly expressed during latency, and is responsible for the replication and maintenance of the viral genome. The expression of LANA is regulated at transcriptional/translational levels through multiple mechanisms, including the secondary structures in the mRNA sequence. LANA mRNA has multiple G-quadruplexes (G4s) that are bound by multiple proteins to stabilize/destabilize these secondary structures for regulating LANA. In this manuscript, we demonstrate the role of Nucleolin (NCL) in regulating LANA expression through its interaction with G-quadruplexes of LANA mRNA. This interaction reduced LANA's protein expression through the sequestration of mRNA into the nucleus, demonstrated by the colocalization of G4-carrying mRNA with NCL. Furthermore, the downregulation of NCL, by way of a short hairpin, showed an increase in LANA translation following an alteration in the levels of LANA mRNA in the cytoplasm. Overall, the data presented in this manuscript showed that G-quadruplexes-mediated translational control could be regulated by NCL, which can be exploited for controlling KSHV latency.


Assuntos
Quadruplex G , Herpesvirus Humano 8 , Humanos , Herpesvirus Humano 8/fisiologia , Nucleolina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Antígenos Virais/genética , Latência Viral/genética
18.
PLoS Pathog ; 19(11): e1011806, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37983265

RESUMO

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs) that target cellular IRFs and/or other innate-immune and stress signaling regulators and suppress the cellular response to viral infection and replication. For vIRF-1, cellular protein targets include IRFs, p53, p53-activating ATM kinase, BH3-only proteins, and antiviral signaling effectors MAVS and STING; vIRF-1 inhibits each, with demonstrated or likely promotion of HHV-8 de novo infection and productive replication. Here, we identify direct interactions of vIRF-1 with STAT3 and STAT-activating Janus kinase TYK2 (the latter reported previously by us to be inhibited by vIRF-1) and suppression by vIRF-1 of cytokine-induced STAT3 activation. Suppression of active, phosphorylated STAT3 (pSTAT3) by vIRF-1 was evident in transfected cells and vIRF-1 ablation in lytically-reactivated recombinant-HHV-8-infected cells led to increased levels of pSTAT3. Using a panel of vIRF-1 deletion variants, regions of vIRF-1 required for interactions with STAT3 and TYK2 were identified, which enabled correlation of STAT3 signaling inhibition by vIRF-1 with TYK2 binding, independently of STAT3 interaction. A viral mutant expressing vIRF-1 deletion-variant Δ198-222 refractory for TYK2 interaction and pSTAT3 suppression was severely compromised for productive replication. Conversely, expression of phosphatase-resistant, protractedly-active STAT3 led to impaired HHV-8 replication. Cells infected with HHV-8 mutants expressing STAT3-refractory vIRF-1 deletion variants or depleted of STAT3 displayed reduced vIRF-1 expression, while custom-peptide-promoted STAT3 interaction could effect increased vIRF-1 expression and enhanced virus replication. Taken together, our data identify vIRF-1 targeting and inhibition of TYK2 as a mechanism of STAT3-signaling suppression and critical for HHV-8 productive replication, the importance of specific pSTAT3 levels for replication, positive roles of STAT3 and vIRF-1-STAT3 interaction in vIRF-1 expression, and significant contributions to lytic replication of STAT3 targeting by vIRF-1.


Assuntos
Herpesvirus Humano 8 , Fator Regulador 1 de Interferon , Humanos , Herpesvirus Humano 8/fisiologia , Fator Regulador 1 de Interferon/metabolismo , Janus Quinases/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteína Supressora de Tumor p53/metabolismo , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Interações Hospedeiro-Patógeno
19.
PLoS Pathog ; 19(11): e1011771, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37934757

RESUMO

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and greater risks of other complications, including malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol(SH)-linked alkylation for the metabolic (SLAM) sequencing and Cleavage Under Target & Release Using Nuclease analysis (CUT&RUN), we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNA polymerase II with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-κB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitors, OTX015 and MZ1, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals.


Assuntos
Infecções por Herpesviridae , Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Interleucina-6/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Citocinas/metabolismo , Infecções por Herpesviridae/metabolismo , Cromatina/metabolismo , Epigênese Genética , Proteínas de Ciclo Celular/metabolismo
20.
Tumour Virus Res ; 16: 200272, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37918513

RESUMO

Approximately 20 % of human cancers are associated with virus infection. DNA tumor viruses can induce tumor formation in host cells by disrupting the cell's DNA replication and repair mechanisms. Specifically, these viruses interfere with the host cell's DNA damage response (DDR), which is a complex network of signaling pathways that is essential for maintaining the integrity of the genome. DNA tumor viruses can disrupt these pathways by expressing oncoproteins that mimic or inhibit various DDR components, thereby promoting genomic instability and tumorigenesis. Recent studies have highlighted the molecular mechanisms by which DNA tumor viruses interact with DDR components, as well as the ways in which these interactions contribute to viral replication and tumorigenesis. Understanding the interplay between DNA tumor viruses and the DDR pathway is critical for developing effective strategies to prevent and treat virally associated cancers. In this review, we discuss the current state of knowledge regarding the mechanisms by which human papillomavirus (HPV), merkel cell polyomavirus (MCPyV), Kaposi's sarcoma-associated herpesvirus (KSHV), and Epstein-Barr virus (EBV) interfere with DDR pathways to facilitate their respective life cycles, and the consequences of such interference on genomic stability and cancer development.


Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 8 , Neoplasias , Humanos , Herpesvirus Humano 4 , Vírus de DNA Tumorais/genética , Neoplasias/genética , Herpesvirus Humano 8/fisiologia , Reparo do DNA/genética , Carcinogênese
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