RESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Like other herpesviruses, it has latent and lytic repertoires. However, there is evidence that some lytic genes can be directly activated by certain cellular factors. Cells undergoing endoplasmic reticulum stress express spliced X-box binding protein 1 (XBP-1s). XBP-1s is also present in large amounts in germinal center B cells. XBP-1s can activate the KSHV replication and transcription activator (RTA) and lytic replication. It can also directly activate KSHV-encoded viral interleukin-6 (vIL-6) and, thus, contribute to the pathogenesis of KSHV MCD. KSHV thymidine kinase (TK), the ORF21 gene product, can enhance the production of dTTP and is important for lytic replication. It can also phosphorylate zidovudine and ganciclovir to toxic moieties, enabling treatment of KSHV-MCD with these drugs. We show here that XBP-1s can directly activate ORF21 and that this activation is mediated primarily through two XBP-response elements (XRE) on the ORF21 promoter region. Deletion or mutation of these elements eliminated XBP-1s-induced upregulation of the promoter, and chromatin immunoprecipitation studies provide evidence that XBP-1s can bind to both XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce ORF21 within 4 hours, and ORF21 expression in the lymph nodes of patients with KSHV-MCD is predominantly found in cells with XBP-1. Thus, XBP-1s may directly upregulate KSHV ORF21 and, thus, contribute to the pathogenesis of KSHV-MCD and the activity of zidovudine and valganciclovir in this disease.IMPORTANCE Spliced X-box binding protein 1 (XBP-1s), part of the unfolded protein response and expressed in developing germinal center B cells, can induce Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and directly activate viral interleukin-6 (vIL-6). We show here that XBP-1s can also directly activate KSHV ORF21, a lytic gene. ORF21 encodes KSHV thymidine kinase (TK), which increases the pool of dTTP for viral replication and enhances lytic replication. Direct activation of ORF21 by XBP-1s can enhance viral replication in germinal center B cells and contribute to the pathogenesis of KSHV multicentric Castleman disease (MCD). KSHV-MCD is characterized by systemic inflammation caused, in part, by lytic replication and overproduction of KSHV vIL-6 in XBP-1s-expressing lymph node plasmablasts. KSHV thymidine kinase can phosphorylate zidovudine and ganciclovir to toxic moieties, and direct activation of ORF21 by XBP-1s may also help explain the effectiveness of zidovudine and valganciclovir in the treatment of KSHV-MCD.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/metabolismo , Timidina Quinase/genética , Proteínas Virais/genética , Proteína 1 de Ligação a X-Box/genética , Hiperplasia do Linfonodo Gigante , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Mutação , Regiões Promotoras Genéticas , Sarcoma de Kaposi/virologia , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Hypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposi's sarcoma (KS) and other AIDS-related malignancies. Kaposi's sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1α and HIF-2α. HIF-1α and HIF-2α have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1α and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1α-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2α in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2α and that both bHLH and PAS domains of HIF-2α interacted with ORF34. Unlike HIF-1α, dose-dependent coexpression of ORF34 stabilized the HIF-2α protein, ensuring HIF-2α-dependent transcriptional activity. The ORF34 protein enhanced HIF-2α ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1α and HIF-2α proteins.IMPORTANCE Hypoxia inducible factor 1α (HIF-1α) and HIF-2α are transcription factors which play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2α, in contrast to its role in binding HIF-1α and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1α and HIF-2α expression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Herpesvirus Humano 8/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Proteínas do Capsídeo/genética , Deleção de Genes , Regulação Viral da Expressão Gênica , Células HEK293 , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Estágios do Ciclo de Vida , Domínios Proteicos , Estabilidade Proteica , Transcrição Gênica , UbiquitinaçãoRESUMO
West Nile Virus (WNV) is endemic in the US and causes severe neurologic disease in horses since its introduction in 1999. There is no effective pharmaceutical treatment for WNV infection rendering vaccination as the only approach to prevention and control of disease. The purpose of this study was to evaluate a recombinant vaccine containing domain III (DIII) of the WNV envelope glycoprotein with and without a natural adjuvant equine (CD40L) in producing virus neutralizing antibodies in horses. Serum IgG1 concentration in the groups of horses vaccinated with the DIII-CD40L+TiterMax and DIII-CD40L proteins were significantly increased (p<0.05) after the second booster vaccination compared to other groups. Serum IgG4 and IgG7, IgG3 and IgG5 concentrations were not significantly increased among all groups. Western blot results showed that animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited the highest specific anti-DIII antibody activities after vaccinations. Moreover, animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited significantly stronger neutralization activity (p<0.05) compared to other groups starting at week eight. The DIII-CD40L protein (with or without TiterMax) stimulated more CD8+T cells, but not CD4+T cells in equine PMBCs. The results demonstrated that vaccination with recombinant WNV E DIII-CD40L protein induced superior humoral and cellular immune response in healthy horses that may be protective against WNV-associated disease in infected animals. CD40L could be utilized as a non-toxic, alternative adjuvant to boost the immunogenicity of subunit vaccines in horses.
Assuntos
Ligante de CD40 , Doenças dos Cavalos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Envelope Viral/imunologia , Febre do Nilo Ocidental/veterinária , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental , Animais , Anticorpos Antivirais/sangue , Ligante de CD40/genética , Ligante de CD40/imunologia , Doenças dos Cavalos/prevenção & controle , Cavalos , Imunoglobulina G/sangue , Testes de Neutralização , Vacinas Sintéticas/imunologia , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/imunologiaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1 beta (IL-1ß) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1ß production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.
Assuntos
Antígenos Virais/metabolismo , Caspase 1/metabolismo , Herpesvirus Humano 8/genética , Linfoma de Efusão Primária/virologia , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/virologia , Latência Viral/fisiologia , Apoptose/genética , Caspase 3/metabolismo , Herpesvirus Humano 8/metabolismo , Interações Hospedeiro-Parasita/fisiologia , HumanosRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761-6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway.
Assuntos
Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Virais/metabolismo , Linhagem Celular , Análise Mutacional de DNA , Humanos , Imunoprecipitação , Ligação Proteica , ProteóliseRESUMO
Hypoxia and hypoxia-inducible factors (HIFs) play an important role in the Kaposi's sarcoma-associated herpesvirus (KSHV) life cycle. In particular, hypoxia can activate lytic replication of KSHV and specific lytic genes, including the replication and transcription activator (RTA), while KSHV infection in turn can increase the levels and activity of HIFs. In the present study, we show that hypoxia increases the levels of mRNAs encoding KSHV latency-associated nuclear antigen (LANA) in primary effusion lymphoma (PEL) cell lines and also increases the levels of LANA protein. Luciferase reporter assays in Hep3B cells revealed a moderate activation of the LANA promoter region by hypoxia as well as by cotransfection with degradation-resistant HIF-1α or HIF-2α expression plasmids. Computer analysis of a 1.2-kb sequence upstream of the LANA translational start site identified six potential hypoxia-responsive elements (HRE). Sequential deletion studies revealed that much of this activity was mediated by one of these HREs (HRE 4R) oriented in the 3' to 5' direction and located between the constitutive (LTc) and RTA-inducible (LTi) mRNA start sites. Site-directed mutation of this HRE substantially reduced the response to both HIF-1α and HIF-2α in a luciferase reporter assay. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated binding of both HIF-1α and HIF-2α to this region. Also, HIF-1α was found to associate with RTA, and HIFs enhanced the activation of LTi by RTA. These results provide evidence that hypoxia and HIFs upregulate both latent and lytic KSHV replication and play a central role in the life cycle of this virus.
Assuntos
Antígenos Virais/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Proteínas Nucleares/genética , Sarcoma de Kaposi/metabolismo , Regulação para Cima , Antígenos Virais/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Herpesvirus Humano 8/metabolismo , Humanos , Hipóxia/genética , Hipóxia/virologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Ativação TranscricionalRESUMO
BACKGROUND: Hypoxia inducible factors (HIFs) are the principal means by which cells upregulate genes in response to hypoxia and certain other stresses. There are two major HIFs, HIF-1 and HIF-2. We previously found that certain genes are preferentially activated by HIF-2. One was protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1). PTPRZ1 is overexpressed in a number of tumors and has been implicated in glioblastoma pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To understand the preferential activation of PTPRZ1 by HIF-2, we studied the PTPRZ1 promoter in HEK293T cells and Hep3B cells. Through deletion and mutational analysis, we identified the principal hypoxia response element. This element bound to both HIF-1 and HIF-2. We further identified a role for ELK1, an E26 transformation-specific (Ets) factor that can bind to HIF-2alpha but not HIF-1alpha, in the HIF-2 responsiveness. Knock-down experiments using siRNA to ELK1 decreased HIF-2 activation by over 50%. Also, a deletion mutation of one of the two Ets binding motifs located near the principal hypoxia response element similarly decreased activation of the PTPRZ1 promoter by HIF-2. Finally, chromatin immunoprecipitation assays showed binding of HIF and ELK1 to the PTPRZ1 promoter region. CONCLUSIONS/SIGNIFICANCE: These results identify HIF-binding and Ets-binding motifs on the PTPRZ1 promoter and provide evidence that preferential activation of PTPRZ1 by HIF-2 results at least in part from cooperative binding of HIF-2 and ELK1 to nearby sites on the PTPRZ1 promoter region. These results may have implications in tumor pathogenesis and in understanding neurobiology, and may help inform the development of novel tumor therapy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Linhagem Celular , Análise Mutacional de DNA , Ativação Enzimática , Genes Reporter , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ligantes , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Primary effusion lymphoma (PEL) is a rare B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). PEL is poorly responsive to standard cytotoxic chemotherapy and portends a poor survival. Consequently, new effective treatment options are urgently needed. It is known that KSHV encodes two lytic genes, ORF36 (phosphotransferase) and KSHV ORF21 (thymidine kinase), which can phosphorylate ganciclovir and azidothymidine, respectively. Here, we have explored whether these genes can be used as therapeutic targets for PEL. PEL arises in pleural spaces and other effusions that provide a hypoxic environment. Based on Northern blot analysis, exposure of PEL cells to hypoxia up-regulated the expression of both ORF36 and ORF21. Using a newly developed nonradioactive reverse-phase high-performance liquid chromatography/mass spectrometry method to separate and quantify the phosphorylated forms of ganciclovir and azidothymidine, we found that PEL cells exposed to hypoxia produced increased amounts of the toxic triphosphates of these drugs. Moreover, we found that hypoxia increased the cell toxicity of ganciclovir and azidothymidine in PEL cells but had no significant effect on the herpesvirus-negative cell line CA46. These findings may have clinical applicability in the development of effective therapies for PEL or other KSHV-related malignancies.
Assuntos
Antivirais/administração & dosagem , Sinergismo Farmacológico , Ganciclovir/administração & dosagem , Herpesviridae/metabolismo , Hipóxia , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/virologia , Zidovudina/administração & dosagem , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Ganciclovir/química , Humanos , Espectrometria de Massas , Fosforilação , Células Tumorais Cultivadas , Zidovudina/químicaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We previously reported that hypoxia activates KSHV lytic replication and that the promoter for open reading frame 34 (ORF34) contains a functional hypoxia-responsive element (HRE). ORF34 is part of a cluster of lytic genes (ORF34-37) that includes ORF36, a phosphotransferase, and ORF37, a shutoff exonuclease. Rapid amplification of cDNA ends analysis revealed that they share a common polyadenylation signal but have two start sites. Two transcripts were identified, one 3.4 kb encoding ORF35-37, and the other 4.2 kb encoding ORF34 and also having coding potential for ORF35-37. Exposure of PEL cell lines to hypoxia induced messages of lengths consistent with those of these transcripts. Reporter assays with Hep3B cells showed activation of both transcripts by hypoxia. The ORF34-37 promoter region has six consensus HREs. Sequential deletion, site-directed mutagenesis experiments, and Northern blot analysis of RNA produced by constructs indicated that the second HRE (HRE-2) plays a critical role in the hypoxic activation of both RNA transcripts. The ORF35-37 transcript was upregulated by cotransfected hypoxia-inducible factor (HIF). Electrophoretic mobility shift assays demonstrated that HRE-2 and ancillary sequences bind and compete for HIF with hypoxic Hep3B nuclear extract. The activation of this gene cluster by hypoxia may have implications for the pathogenesis of PEL and KS. Moreover, the activation of ORF36 by hypoxia might be exploited to develop targeted therapy for PEL, which arises in a hypoxic environment (pleural effusions).
Assuntos
Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 8/fisiologia , Família Multigênica/genética , Fases de Leitura Aberta/genética , Elementos de Resposta/genética , Ativação Viral/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/genética , Linhagem Celular , Sistema Livre de Células , Exonucleases/genética , Humanos , Linfoma/genética , Linfoma/virologia , Mutagênese Sítio-Dirigida , Fosfotransferases/genética , RNA Mensageiro/genética , RNA Viral/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Deleção de Sequência , Transcrição Gênica/genética , Regulação para Cima/genética , Proteínas Virais/genéticaRESUMO
Cells exposed to hypoxia respond by increasing the level of hypoxia-inducible factor-1 (HIF-1). This factor then activates a number of genes by binding to hypoxia response elements in their promoter regions. A second hypoxia-responsive factor, HIF-2, can activate many of the same genes as HIF-1. Overexpression of HIFs accompanies the pathogenesis of many tumors. It is unclear, however, as to the respective role of these factors in responsiveness to hypoxia and other stresses. To address this issue, we used microarray technology to study the genes activated in HEK293T cells by hypoxia or transfection with the alpha chain of HIF-1 (or mutant HIF-1 resistant to degradation) or HIF-2. Fifty-six genes were found to be up-regulated at least 3-fold by either hypoxia or transfection. Of these, 21 were elevated both by transfection with HIF-1alpha and with HIF-2alpha, and 14 were preferentially activated by HIF-1alpha including several involved in glycolysis. Ten genes were preferentially activated by HIF-2alpha, including two (CACNA1A and PTPRZ1) implicated in neurologic diseases. Interestingly, most HIF-2alpha-responsive genes were not substantially activated by hypoxia. An additional 10 genes were up-regulated by hypoxia but minimally activated by HIF-1alpha or HIF-2alpha transfection. Ten of the genes were studied by quantitative real-time PCR and/or by Northern blot and the results paralleled those found with microarray technology. Although confirmation in other systems will be necessary, these results indicate that whereas some genes are robustly activated by both HIF-1 and HIF-2, others can be preferentially activated by one or the other factor.
Assuntos
Transativadores/genética , Fatores de Transcrição/genética , Regulação para Cima/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Northern Blotting , Hipóxia Celular/genética , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Rim/citologia , Rim/metabolismo , Rim/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/biossíntese , Fatores de Transcrição/biossíntese , TransfecçãoRESUMO
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is an etiologic agent of KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease. We recently demonstrated that hypoxia can induce lytic replication of KSHV in PEL cell lines. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF), and we hypothesized that the KSHV genome may respond to hypoxia through functional hypoxia response elements (HREs). Here, we demonstrate the presence of at least two promoters within the KSHV genome that are activated by hypoxia or hypoxia mimics. One is in the promoter region of the gene for Rta, the main lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three putative consensus HREs oriented in the direction of the gene. Dissection and site-directed mutagenesis studies confirmed that one of the HREs of the ORF34 promoter is functional. Under conditions of hypoxia, the ORF34 promoter was strongly upregulated by HIF-1 alpha and HIF-2 alpha. By contrast, the promoter of the gene for Rta appeared to be preferentially upregulated by HIF-2 alpha. Reverse transcription-PCR analysis revealed that specific messages for ORF34 and ORF50 are upregulated in BCBL-1 cells exposed to hypoxia. An HIF-1 binding and competition assay demonstrated that the HRE sequence from the ORF34 promoter can compete for HIF-1 alpha binding to an erythropoietin HRE oligonucleotide while a mutant sequence cannot. Thus, we demonstrated that a viral gene can be activated by hypoxia through activation of a functional viral HRE. To our knowledge, this is the first example of a functional HRE in a viral promoter.
Assuntos
Hipóxia Celular , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Elementos de Resposta/genética , Ativação Viral , Ligação Competitiva , Linhagem Celular , Eritropoetina/metabolismo , Herpesvirus Humano 8/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta/genética , Fases de Leitura Aberta/fisiologia , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Elementos de Resposta/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The expression of major histocompatibility complex class I (MHC-I) molecules at the cell surface was down-regulated in BC-3 cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 at early times after treatment with 12-O-tetradecanoylphorbol acetate (TPA), and in HeLa cells transfected with the K5 gene of KSHV. However, an immunoprecipitation study on these cells with anti-MHC-I monoclonal antibody revealed that there was no significant reduction in the synthesis of MHC-I molecules. A pulse-chase analysis followed by endoglycosidase H digestion also demonstrated the stability and transport of MHC-I molecules from the endoplasmic reticulum to at least the medial-GOLGI: K5 antigen was clearly detected by immunohistological examination of samples from Kaposi's sarcoma, primary effusion lymphoma and Castleman's disease. These results suggest that the down-regulation of MHC-I molecules by K5 gene expression during reactivation may be important for evading immunological surveillance in the host.