RESUMO
Kaposi sarcoma (KS) is a neoplasm of vascular origin that promotes angiogenesis and the growth of endothelial cells triggered by the Kaposi Sarcoma-associated Herpes Virus (KSHV). When associated with HIV, KSHV becomes more aggressive and rapidly evolves. The HIV-1 TAT protein can be essential in developing AIDS-associated KS by promoting angiogenesis and increasing KSHV replication. Therefore, we evaluated the genetic profile of the first exon of tat gene among groups of people living with HIV (PLHIV) with (case group, n = 36) or without KS, this later with (positive control group, n = 46) and without KSHV infection (negative control group, n = 24); all individuals under antiretroviral therapy. The genetic diversity, the DN/DS ratio, and the genetic entropy of the first exon of tat were higher in the case group, followed by the positive control group, which was higher than the negative control group. The number of tat codons under positive selection was seven in the case group, six in the positive control group, and one in the negative control group. The prevalence of HIV viral loads below the detection limit was equal in the case and positive control groups, which were lower than in the negative control group. The mean CD4+ T cell counts were higher in the negative control group, followed by the positive control group, and followed by the case group. These results emphasize the negative influence of KSHV in antiretroviral treatment, as well as the HIV-specific TAT profile among PLHIV who developed KS.
Assuntos
Coinfecção , Infecções por HIV , Herpesvirus Humano 8 , Sarcoma de Kaposi , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , Sarcoma de Kaposi/virologia , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Masculino , Herpesvirus Humano 8/genética , Feminino , Adulto , Pessoa de Meia-Idade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Coinfecção/virologia , Coinfecção/tratamento farmacológico , HIV-1/genética , HIV-1/efeitos dos fármacos , Variação Genética , Carga Viral , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4RESUMO
Primary effusion lymphoma (PEL) is a malignant B-cell lymphoma attributable to Kaposi sarcoma-associated herpesvirus (KSHV) infection. PEL is characterized by invasive behavior, showing recurrent effusions in body cavities. The clinical outcome and typical prognosis in patients with PEL are poor and potentially lethal. Clarification of the pathogenesis in PEL is urgently needed in order to develop novel therapies. PEL cells generally lack B-cell surface markers, and we therefore hypothesized that the B-cell transcription factor, PAX5, would be down-regulated in PEL. The expression of PAX5 is detected from the pro-B to the mature B-cell stage and is indispensable for the differentiation of B-cells. PAX5 was silenced in PEL cells via its promoter methylation. Up-regulation of PAX5 induced several genes coding for B-cell surface marker mRNA, but not protein level. PAX5 inhibited cell growth via G1 cell cycle arrest. PAX5 bound to RB and increased its protein expression. RB/E2F-regulated genes were significantly down-regulated in microarray analysis and PCR experiments. To elucidate the in vivo role of PAX5, we examined the restoration of PAX5 in a PEL mouse model. The ascites volume and organ invasions were significantly suppressed by PAX5 restoration. Reduction of PAX5 has played a crucial role in the oncogenesis of PEL, and PAX5 is a tumor suppressor in PEL. Targeting PAX5 could represent a novel therapeutic strategy for patients with PEL.
Assuntos
Pontos de Checagem do Ciclo Celular , Herpesvirus Humano 8 , Linfoma de Efusão Primária , Fator de Transcrição PAX5 , Fator de Transcrição PAX5/metabolismo , Fator de Transcrição PAX5/genética , Linfoma de Efusão Primária/virologia , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/etiologia , Animais , Humanos , Herpesvirus Humano 8/genética , Camundongos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Proliferação de Células , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Regiões Promotoras Genéticas , Modelos Animais de DoençasRESUMO
OBJECTIVES: Human herpesvirus 8 (HHV8) is rarely studied in Congo, despite its prevalence in Africa. Among healthy individuals, HHV-8 does not always lead to a life-threatening infection; however, in immunocompromised individuals, it could lead to more severe disease. The distribution of HHV-8 genotypes varies depending on ethnicity and geographic region. METHOD: A prospective cross-sectional study included 265 samples from healthy blood donors from the National Blood Transfusion Center in Brazzaville, with an average age of 35 years, with extremes ranging from 18 to 60 years. After DNA extraction, a nested PCR was carried out for molecular detection, followed by genotyping by amplification of specific primers. RESULT: In this study, 4.9% were positive for molecular detection of HHV-8 DNA. All HHV-8 positive DNA samples that were subjected to genotyping by amplification with specific primers allowing discrimination of two major genotypes (A and B). Genotype A was identified in 5 (1.9%) samples and genotype B in 2 (0.7%) samples, indicating that both genotypes were predominant. The remaining viral DNA samples not identified as the major genotypes were classified as «indeterminate¼ and consisted of 6 (2.3%) samples. CONCLUSION: The results of the study suggest that Congo is an area where HHV-8 infection is endemic.
Assuntos
Doadores de Sangue , DNA Viral , Genótipo , Infecções por Herpesviridae , Herpesvirus Humano 8 , Humanos , Congo/epidemiologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/isolamento & purificação , Herpesvirus Humano 8/classificação , Adulto , Masculino , Feminino , Pessoa de Meia-Idade , DNA Viral/genética , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/sangue , Adolescente , Estudos Transversais , Estudos Prospectivos , Reação em Cadeia da PolimeraseRESUMO
Recently published near full-length KSHV genomes from a Cameroon Kaposi sarcoma case-control study showed strong evidence of viral recombination and mixed infections, but no sequence variations associated with disease. Using the same methodology, an additional 102 KSHV genomes from 76 individuals with KSHV-associated diseases have been sequenced. Diagnoses comprise all KSHV-associated diseases (KAD): Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated large cell lymphoma (KSHV-LCL), a type of multicentric Castleman disease (KSHV-MCD), and KSHV inflammatory cytokine syndrome (KICS). Participants originated from 22 different countries, providing the opportunity to obtain new near full-length sequences of a wide diversity of KSHV genomes. These include near full-length sequence of genomes with KSHV K1 subtypes A, B, C, and F as well as subtype E, for which no full sequence was previously available. High levels of recombination were observed. Fourteen individuals (18%) showed evidence of infection with multiple KSHV variants (from two to four unique genomes). Twenty-six comparisons of sequences, obtained from various sampling sites including PBMC, tissue biopsies, oral fluids, and effusions in the same participants, identified near complete genome conservation between different biological compartments. Polymorphisms were identified in coding and non-coding regions, including indels in the K3 and K15 genes and sequence inversions here reported for the first time. One such polymorphism in KSHV ORF46, specific to the KSHV K1 subtype E2, encoded a mutation in the leucine loop extension of the uracil DNA glycosylase that results in alteration of biochemical functions of this protein. This confirms that KSHV sequence variations can have functional consequences warranting further investigation. This study represents the largest and most diverse analysis of KSHV genome sequences to date among individuals with KAD and provides important new information on global KSHV genomics.
Assuntos
Genoma Viral , Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/genética , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Polimorfismo Genético , Idoso , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Etnicidade/genética , Hiperplasia do Linfonodo Gigante/virologia , Hiperplasia do Linfonodo Gigante/genética , FilogeniaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.
Assuntos
Herpesvirus Humano 8 , Canal de Potássio Kv1.3 , Fatores de Transcrição NFATC , Replicação Viral , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/antagonistas & inibidores , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Transativadores/metabolismo , Transativadores/genética , Linfócitos B/virologia , Linfócitos B/metabolismo , Cálcio/metabolismo , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/genéticaRESUMO
Like many other viruses, KSHV has two life cycle modes: the latent phase and the lytic phase. The RTA protein from KSHV is essential for lytic reactivation, but how this protein's activity is regulated is not fully understood. Here, we report that linear ubiquitination regulates the activity of RTA during KSHV lytic reactivation and de novo infection. Overexpressing OTULIN inhibits KSHV lytic reactivation, whereas knocking down OTULIN or overexpressing HOIP enhances it. Intriguingly, we found that RTA is linearly polyubiquitinated by HOIP at K516 and K518, and these modifications control the RTA's nuclear localization. OTULIN removes linear polyubiquitin chains from cytoplasmic RTA, preventing its nuclear import. The RTA orthologs encoded by the EB and MHV68 viruses are also linearly polyubiquitinated and regulated by OTULIN. Our study establishes that linear polyubiquitination plays a critically regulatory role in herpesvirus infection, adding virus infection to the list of biological processes known to be controlled by linear polyubiquitination.
Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Transativadores , Ubiquitinação , Replicação Viral , Herpesvirus Humano 8/fisiologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/genética , Células HEK293 , Transativadores/metabolismo , Transativadores/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ativação Viral , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Núcleo Celular/metabolismoRESUMO
Primary effusion lymphoma (PEL) is a rare B-cell non-Hodgkin lymphoma associated with Kaposi Sarcoma-associated herpesvirus (KSHV/HHV8) infection. Lymphoma cells are coinfected with Epstein-Barr virus (EBV) in 60-80% of cases. Tools allowing a reliable PEL diagnosis are lacking. This study reports PEL diagnosis in 4 patients using a Flow-Fluorescence in situ hybridization (FlowFISH) technique that allowed detection of differentially expressed EBV and HHV8 transcripts within the same sample, revealing viral heterogeneity of the disease. Moreover, infected cells exhibited variable expressions of CD19, CD38, CD40, and CD138. Therefore, FlowFISH is a promising tool to diagnose and characterize complex viral lymphoproliferations.
Assuntos
Herpesvirus Humano 4 , Herpesvirus Humano 8 , Hibridização in Situ Fluorescente , Linfoma de Efusão Primária , Humanos , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/isolamento & purificação , Hibridização in Situ Fluorescente/métodos , Linfoma de Efusão Primária/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Masculino , Idoso , Pessoa de Meia-Idade , Feminino , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/complicações , Idoso de 80 Anos ou maisRESUMO
The viral interferon regulatory factors (vIRFs) of KSHV are known to dysregulate cell signaling pathways to promote viral oncogenesis and to block antiviral immune responses to facilitate infection. However, it remains unknown to what extent each vIRF plays a role in gene regulation. To address this, we performed a comparative analysis of the protein structures and gene regulation of the four vIRFs. Our structure prediction analysis revealed that despite their low amino acid sequence similarity, vIRFs exhibit high structural homology in both their DNA-binding domain (DBD) and IRF association domain. However, despite this shared structural homology, we demonstrate that each vIRF regulates a distinct set of KSHV gene promoters and human genes in epithelial cells. We also found that the DBD of vIRF1 is essential in regulating the expression of its target genes. We propose that the structurally similar vIRFs evolved to possess specialized transcriptional functions to regulate specific genes.
Assuntos
Células Epiteliais , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8 , Fatores Reguladores de Interferon , Proteínas Virais , Humanos , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Células Epiteliais/virologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Genoma Viral , Linhagem CelularRESUMO
Kaposi's sarcoma-associated herpesvirus is the etiologic agent of Kaposi's sarcoma and two B-cell malignancies. Recent advancements in sequencing technologies have led to high resolution transcriptomes for several human herpesviruses that densely encode genes on both strands. However, for KSHV progress remained limited due to the overall low percentage of KSHV transcripts, even during lytic replication. To address this challenge, we have developed a target enrichment method to increase the KSHV-specific reads for both short- and long-read sequencing platforms. Furthermore, we combined this approach with the Transcriptome Resolution through Integration of Multi-platform Data (TRIMD) pipeline developed previously to annotate transcript structures. TRIMD first builds a scaffold based on long-read sequencing and validates each transcript feature with supporting evidence from Illumina RNA-Seq and deepCAGE sequencing data. Our stringent innovative approach identified 994 unique KSHV transcripts, thus providing the first high-density KSHV lytic transcriptome. We describe a plethora of novel coding and non-coding KSHV transcript isoforms with alternative untranslated regions, splice junctions and open-reading frames, thus providing deeper insights on gene expression regulation of KSHV. Interestingly, as described for Epstein-Barr virus, we identified transcription start sites that augment long-range transcription and may increase the number of latency-associated genes potentially expressed in KS tumors.
Assuntos
Processamento Alternativo , Herpesvirus Humano 8 , Transcriptoma , Herpesvirus Humano 8/genética , Humanos , Transcriptoma/genética , Transcrição Gênica , Regulação Viral da Expressão Gênica , Fases de Leitura Aberta/genética , Sequenciamento de Nucleotídeos em Larga Escala , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/genética , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus with a double-stranded DNA capable of establishing latent infection in the host cell. During latency, only a limited number of viral genes are expressed in infected host cells, and that helps the virus to evade host immune cell response. During primary infection, the KSHV genome is chromatinized and maintained as an episome, which is tethered to the host chromosome via Latency Associated Nuclear Antigen (LANA). The KSHV episome undergoes the same chromatin modification with the host cell chromosome and, therefore, is regulated by various epigenetic modifications, such as DNA methylation, histone methylation, and histone acetylation. The KSHV genome is also organized in a spatiotemporal manner by forming genomic loops, which enable simultaneous and coordinated control of dynamic gene transcription, particularly during the lytic replication phase. The genome-wide approaches and advancing bioinformatic tools have increased the resolution of studies on the dynamic transcriptional control and our understanding of KSHV latency-lytic switch regulation. We will summarize our current understanding of the epigenetic gene regulation on the KSHV chromatin.
Assuntos
Cromatina , Epigênese Genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8 , Ativação Viral , Latência Viral , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Humanos , Cromatina/metabolismo , Cromatina/genética , Latência Viral/genética , Genoma Viral , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/genética , Interações Hospedeiro-Patógeno , Antígenos Virais , Proteínas NuclearesRESUMO
Kaposi's sarcoma (KS) is a cancer affecting skin and internal organs for which the Kaposi's sarcoma associated herpesvirus (KSHV) is a necessary cause. Previous work has pursued KS diagnosis by quantifying KSHV DNA in skin biopsies using a point-of-care (POC) device which performs quantitative loop-mediated isothermal amplification (LAMP). These previous studies revealed that extracting DNA from patient biopsies was the rate limiting step in an otherwise rapid process. In this study, a simplified, POC-compatible alkaline DNA extraction, ColdSHOT, was optimized for 0.75 mm human skin punch biopsies. The optimized ColdSHOT extraction consistently produced 40,000+ copies of DNA per 5 µl reaction from 3 mg samples-a yield comparable to standard spin column extractions-within 1 h without significant equipment. The DNA yield was estimated sufficient for KSHV detection from KS-positive patient biopsies, and the LAMP assay was not affected by non-target tissue in the unpurified samples. Furthermore, the yields achieved via ColdSHOT were robust to sample storage in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer prior to DNA extraction, and the DNA sample was stable after extraction. The results presented in this study indicate that the ColdSHOT DNA extraction could be implemented to simplify and accelerate the LAMP-based diagnosis of Kaposi's sarcoma using submillimeter biopsy samples.
Assuntos
DNA Viral , Herpesvirus Humano 8 , Técnicas de Amplificação de Ácido Nucleico , Sarcoma de Kaposi , Pele , Humanos , DNA Viral/genética , DNA Viral/isolamento & purificação , Herpesvirus Humano 8/isolamento & purificação , Herpesvirus Humano 8/genética , Biópsia/métodos , Pele/virologia , Pele/patologia , Sarcoma de Kaposi/diagnóstico , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Técnicas de Diagnóstico Molecular/métodosRESUMO
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/virologiaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.
Assuntos
Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 8 , Regiões Promotoras Genéticas , Ativação Viral , Latência Viral , Humanos , Antígenos Virais/genética , Antígenos Virais/metabolismo , Cromatina/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Células HEK293 , Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sequências Repetidas Terminais/genética , Transativadores/metabolismo , Ativação Viral/genética , Latência Viral/genéticaRESUMO
Human Herpesvirus 8 (HHV-8) has been classified by sequence analysis of open reading frame (ORF) K1, ORF K15, and variable sequence loci within the central constant region. The purpose of this study was to examine the molecular epidemiology of HHV-8 in an Irish population. This retrospective study included 30 patients who had HHV-8 DNA detected in plasma. Nested end-point PCR was used to characterise four regions of the HHV-8 genome, K1, T0.7 (K12), ORF 75, and K15. Sequencing data were obtained for 23 specimens from 19 patients. Phylogenetic analysis of ORF K1 demonstrated that subtypes A, B, C and F were present in 37%, 11%, 47% and 5%, respectively. For T0.7 and ORF 75, sequencing data were obtained for 12 patients. For T0.7, subtypes A/C, J, B, R and Q were present in 58%, 17%, 8%, 8%, and 8%, respectively. For ORF 75, subtypes A, B, C and D were present in 58%, 8%, 25%, and 8%, respectively. K15 sequences were determined for 13 patients. 69% had the P allele and 31% had the M allele. The data generated by this study demonstrate that a broad variety of HHV-8 subtypes are represented in patients exhibiting HHV-8-related disease in Ireland, a low prevalence country. The predominance of C and A K1 subtypes was as expected for a Western European population. The 31% prevalence for K15 subtype M was higher than expected for a Western European population. This may represent the changing and evolving epidemiology in Ireland due to altered migration patterns.
Assuntos
DNA Viral , Infecções por Herpesviridae , Herpesvirus Humano 8 , Epidemiologia Molecular , Filogenia , Análise de Sequência de DNA , Humanos , Irlanda/epidemiologia , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/classificação , Herpesvirus Humano 8/isolamento & purificação , Masculino , Feminino , Estudos Retrospectivos , Pessoa de Meia-Idade , Adulto , DNA Viral/genética , Idoso , Adulto Jovem , Reação em Cadeia da Polimerase , Genótipo , Adolescente , Fases de Leitura Aberta , Idoso de 80 Anos ou mais , Criança , Dados de Sequência MolecularRESUMO
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 , FosfoproteínasRESUMO
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 CélulasRESUMO
Kaposi sarcoma (KS) is the most common cancer in persons living with HIV. It is caused by KS-associated herpesvirus (KSHV). There exists no animal model for KS. Pronuclear injection of the 170,000-bp viral genome induces early-onset, aggressive angiosarcoma in transgenic mice. The tumors are histopathologically indistinguishable from human KS. As in human KS, all tumor cells express the viral latency-associated nuclear antigen (LANA). The tumors transcribe most viral genes, whereas endothelial cells in other organs only transcribe the viral latent genes. The tumor cells are of endothelial lineage and exhibit the same molecular pattern of pathway activation as KS, namely phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR, interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF). The KSHV-induced tumors are more aggressive than Ha-ras-induced angiosarcomas. Overall survival is increased by prophylactic ganciclovir. Thus, whole-virus KSHV-transgenic mice represent an accurate model for KS and open the door for the genetic dissection of KS pathogenesis and evaluation of therapies, including vaccines.
Assuntos
Modelos Animais de Doenças , Hemangiossarcoma , Herpesvirus Humano 8 , Camundongos Transgênicos , Sarcoma de Kaposi , Animais , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidade , Camundongos , Hemangiossarcoma/virologia , Hemangiossarcoma/genética , Hemangiossarcoma/patologia , Sarcoma de Kaposi/virologia , Sarcoma de Kaposi/patologia , Genoma Viral , Humanos , Antígenos Virais/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Ganciclovir/uso terapêutico , Ganciclovir/farmacologia , Interleucina-10/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The tegument is a structure that is unique to herpesviruses that includes host and viral proteins, including the viral ORF42 and ORF55 proteins. Alphaherpesvirus tegument proteins have been well studied, but much is unknown regarding KSHV. Here, we report an interaction between the ORF42 and ORF55 proteins. ORF55 interacted with and recruited ORF42 from the nucleus to the cytoplasm. When ORF42 and ORF55 were expressed simultaneously in cultured cells, the expression level of these two viral proteins was higher than when either was expressed independently. ORF55, but not ORF42, was polyubiquitinated, suggesting that an unidentified regulatory mechanism may be present. A recombinant virus with an ectopic stop codon in ORF42 exhibited normal replication of genomic DNA, but fewer virus particles were released with the recombinant than with the wild-type virus. A unique R136Q mutation in ORF42, which is found in a KSHV strain that is prevalent on Miyako Island, Okinawa Prefecture, Japan, further increased the expression of ORF42 and ORF55 when these proteins were expressed simultaneously. However, the ORF42 R136Q mutation did not affect the localization pattern of ORF42 itself or of ORF55. In addition, experiments with a recombinant virus possessing the ORF42 R136Q mutation showed lower levels of production of the mutant virus than of the wild-type virus, despite similar levels of genome replication. We suggest that the R136Q mutation in ORF42 plays an important role in ORF55 protein expression and virus production.
Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/genética , Citoplasma , Japão , Proteínas Virais/genéticaRESUMO
Human herpesvirus 8 (HHV8)-associated diseases include Kaposi sarcoma (KS), multicentric Castleman disease (MCD), germinotropic lymphoproliferative disorder (GLPD), Kaposi sarcoma inflammatory cytokine syndrome (KICS), HHV8-positive diffuse large B-cell lymphoma (HHV8+ DLBCL), primary effusion lymphoma (PEL), and extra-cavitary PEL (ECPEL). We report the case of a human immunodeficiency virus (HIV)-negative male treated for cutaneous KS, who developed generalized lymphadenopathy, hepatosplenomegaly, pleural and abdominal effusions, renal insufficiency, and pancytopenia. The excised lymph node showed features of concomitant involvement by micro-KS and MCD, with aggregates of HHV8+, Epstein Barr virus (EBV)-negative, IgM+, and lambda+ plasmablasts reminiscent of microlymphoma. Molecular investigations revealed a somatically hypermutated (SHM) monoclonal rearrangement of the immunoglobulin heavy chain (IGH), accounting for 4% of the B-cell population of the lymph node. Mutational analyses identified a pathogenic variant of KMT2D and variants of unknown significance in KMT2D, FOXO1, ARID1A, and KMT2A. The patient died shortly after surgery. The histological features (HHV8+, EBV-, IgM+, Lambda+, MCD+), integrated with the molecular findings (monoclonal IGH, SHM+, KMT2D mutated), supported the diagnosis of a monoclonal HHV8+ microlymphoma, with features intermediate between an incipient HHV8+ DLBCL and an EBV-negative ECPEL highlighting the challenges in the accurate classification of HHV8-driven lymphoid proliferations.
Assuntos
Hiperplasia do Linfonodo Gigante , Infecções por Vírus Epstein-Barr , Infecções por HIV , Herpesvirus Humano 8 , Sarcoma de Kaposi , Masculino , Humanos , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/genética , Herpesvirus Humano 4 , Infecções por HIV/complicações , Imunoglobulina MRESUMO
Ferroptosis, a defensive strategy commonly employed by the host cells to restrict pathogenic infections, has been implicated in the development and therapeutic responses of various types of cancer. However, the role of ferroptosis in oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-induced cancers remains elusive. While a growing number of non-histone proteins have been identified as acetylation targets, the functions of these modifications have yet to be revealed. Here, we show KSHV reprogramming of host acetylation proteomics following cellular transformation of rat primary mesenchymal precursor. Among them, SERPINE1 mRNA binding protein 1 (SERBP1) deacetylation is increased and required for KSHV-induced cellular transformation. Mechanistically, KSHV-encoded viral interleukin-6 (vIL-6) promotes SIRT3 deacetylation of SERBP1, preventing its binding to and protection of lipoyltransferase 2 (Lipt2) mRNA from mRNA degradation resulting in ferroptosis. Consequently, a SIRT3-specific inhibitor, 3-TYP, suppresses KSHV-induced cellular transformation by inducing ferroptosis. Our findings unveil novel roles of vIL-6 and SERBP1 deacetylation in regulating ferroptosis and KSHV-induced cellular transformation, and establish the vIL-6-SIRT3-SERBP1-ferroptosis pathways as a potential new therapeutic target for KSHV-associated cancers.