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Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms.

ABSTRACT: UNLABELLED:Kaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1 (HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitro proliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellular HO-1 transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, the BACH1 message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation of HO-1 and BACH1 during viral latency. Interestingly, the data indicate that neither miR-K12-11 nor de novo KSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype. IMPORTANCE:While the mechanisms underlying KSHV induction of HO-1 remain unknown, the cellular mechanisms that regulate HO-1 expression have been extensively investigated in the context of basal and pathophysiological states. The detoxifying action of HO-1 is critical for the protection of cells exposed to high heme levels. KS spindle cells are erythrophagocytic and contain erythrocyte ghosts. Erythrocyte degeneration leads to the localized release of heme, creating oxidative stress that may be further exacerbated by environmental or other cofactors. Our previous work showed that KSHV-infected cells proliferate in response to heme and that this occurs in a HO-1-dependent manner. We therefore hypothesize that KSHV induction of HO-1 contributes to KS tumor development via heme metabolism and propose that HO-1 be evaluated as a therapeutic target for KS. Our present work, which aimed to understand the mechanisms whereby KSHV induces HO-1, will be important for the design and implementation of such a strategy.

SUBMITTER: Botto S

PROVIDER: S-EPMC4462627 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Project description:Intra-host tumor virus variants may influence the pathogenesis and treatment responses of some virally-associated cancers. However, the intra-host variability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma (KS), has to date been explored with sequencing technologies that possibly introduce more errors than that which occurs in the viral population, and these studies have only studied variable regions. Here, full-length KSHV genomes in tumors and/or oral swabs from 9 Ugandan adults with HIV-associated KS were characterized. Furthermore, we used deep, short-read sequencing using duplex unique molecular identifiers (dUMI)-random double-stranded oligonucleotides that barcode individual DNA molecules before library amplification. This allowed suppression of PCR and sequencing errors to ~10-9/base as well as afforded accurate determination of KSHV genome numbers sequenced in each sample. KSHV genomes were assembled de novo, and rearrangements observed were confirmed by PCR and Sanger sequencing. 131-kb KSHV genome sequences, excluding major repeat regions, were successfully obtained from 23 clinical specimens, averaging 2.3x104 reads/base. Strikingly, KSHV genomes were virtually identical within individuals at the point mutational level. The intra-host heterogeneity that was observed was confined to tumor-associated KSHV mutations and genome rearrangements, all impacting protein-coding sequences. Although it is unclear whether these changes were important to tumorigenesis or occurred as a result of genomic instability in tumors, similar changes were observed across individuals. These included inactivation of the K8.1 gene in tumors of 3 individuals and retention of a region around the first major internal repeat (IR1) in all instances of genomic deletions and rearrangements. Notably, the same breakpoint junctions were found in distinct tumors within single individuals, suggesting metastatic spread of rearranged KSHV genomes. These findings define KSHV intra-host heterogeneity in vivo with greater precision than has been possible in the past and suggest the possibility that aberrant KSHV genomes may contribute to aspects of KS tumorigenesis. Furthermore, study of KSHV with use of dUMI provides a proof of concept for utilizing this technique for detailed study of other virus populations in vivo.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. Immediately after infection, KSHV transcribes many lytic genes that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, microRNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virion packaged transcripts and the profiles of viral genes transcribed after de novo infections of various cell types (human peripheral blood mononuclear cells [PBMCs], CD14(+) monocytes, and telomerase-immortalized vascular endothelial [TIVE] cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, open reading frame 58 [ORF58], ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines, with the majority of the genes, other than latent genes, silencing after 24 h postinfection. We differentiated the actively transcribing genes from the virion-transduced transcripts using a nascent RNA capture approach (Click-iT chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA) to block the activity of viral DNA polymerase confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performed de novo PBMC infections with a recombinant ORF59-deleted KSHV virus, which showed significantly reduced numbers of viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency.Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. KSHV establishes a lifelong latency in the infected host, during which only a limited number of viral genes are expressed. However, a fraction of latently infected cells undergo spontaneous reactivation to produce virions that infect the surrounding cells. These newly infected cells are primed early to retain the incoming viral genome and induce cell growth. KSHV transcribes a variety of lytic proteins during de novo infections that modulate various cellular pathways to establish the latent infection. Interestingly, a large number of viral proteins and RNA are encapsidated in the infectious virions and transduced into the infected cells during a de novo infection. This study determined the kinetics of the viral gene expression during de novo KSHV infections and the functional role of the incoming viral transcripts in establishing latency.

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Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms.

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