Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during
            <i>De Novo</i>
            Primary Infection of Human B and Endothelial Cells

Activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway has been associated with numerous human malignancies, including primary effusion lymphomas (PELs). PEL, a cancerous proliferation of B cells, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Previously we identified constitutive phosphorylation of STAT6 on tyrosine 641 (p-STAT6C ) in PEL cell lines BC3 and BCBL1; however, the molecular mechanism leading to this activation remains unclear. Here we demonstrate that STAT6 activation tightly correlates with interleukin-13 (IL-13) secretion, JAK1/2 tyrosine phosphorylation, and reduced expression of SHP1 due to KSHV infection. Moreover, p-STAT6 C and reduction of SHP1 were also observed in KS patient tissue. Notably, blockade of IL-13 by antibody neutralization dramatically inhibits PEL cell proliferation and survival. Taken together, these results suggest that IL-13/STAT6 signaling is modulated by KSHV to promote host cell proliferation and viral pathogenesis. IMPORTANCESTAT6 is a member of signal transducer and activator of transcription (STAT) family, whose activation is linked to KSHV-associated cancers. The mechanism through which STAT6 is modulated by KSHV remains unclear. In this study, we demonstrated that constitutive activation of STAT6 in KSHV-associated PEL cells results from interleukin-13 (IL-13) secretion and reduced expression of SHP1. Importantly, we also found that depletion of IL-13 reduces PEL cell growth and survival. This discovery provides new insight that IL-13/STAT6 plays an essential role in KSHV pathogenesis. Cytokines play a critical role in many viral infections. Viruses not only manipulate host cytokine production to favor virus survival, replication, and infection but also help virus-infected cells to modulate the host immune response, which potentially results in the development of viral persistent infection, pathogenesis, or tumorigenesis (1). Kaposi's sarcoma-associated herpesvirus (KSHV), also named human herpesvirus 8 (HHV-8), is an oncogenic gammaherpesvirus that associates with several aggressive malignancies, including AIDS-related Kaposi's sarcoma (KS) (2), primary effusion lymphoma (PEL) (3), and multicentric Castleman's disease (MCD) (4). Increasing evidence has suggested that KSHV also deregulates an array of host cytokines, including interleukin-6 (IL-6), IL-8, and IL-1␣, thereby inducing cell proliferation and malignant transformation (5-8).Signal transducer and activator of transcription (STAT) proteins are a family of cytoplasmic transcription factors involved in cytokine signal transduction. STAT6 is a key member of the STAT family, whose role in the biology of cancer and immune cells has been firmly established (9, 10). STAT6 is activated by cytokine IL-4 or IL-13, via a common receptor chain, namely, IL-4R␣. Upon interleukin binding, IL-4R␣ dimerizes with IL-4R␥ or IL-13R␣1 to form type I or type II IL-4R receptor, respectively. The dimerized receptor recruits and activates phosphorylatio...

Section: Discussionmentioning

confidence: 99%

Section: Kshv Induces Constitutive Phosphorylation Of Stat6 (P-stat6 mentioning

confidence: 99%

Constitutive Activation of Interleukin-13/STAT6 Contributes to Kaposi's Sarcoma-Associated Herpesvirus-Related Primary Effusion Lymphoma Cell Proliferation and Survival

Wang

Zhu

Wei

et al. 2015

“…In support of the potential to produce small peptides (Ͻ100 amino acids), a portion of T1.5 is found in the cytoplasm by Northern blotting and by RNA fluorescence in situ hybridization (FISH) (21), whereas T3.0 produces at least three small peptides (41), one of which induces its antisense transcript RTA (43). PAN RNA and T1.5 are also packaged within virions and expressed in the early stages of primary infection, suggesting an important role in establishing a successful infection (35,36). Two other lncRNAs, ALT (antisense-to-latency transcripts) and T6.1, have been consistently observed in transcriptome sequencing (RNA-seq) surveys.…”

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confidence: 99%

“…Its origin and regulation has been well characterized (22,(25)(26)(27)(28)(29)(30)(31)(32). In situ studies showed that PAN RNA is either not expressed or expressed at low basal levels during latency (33,34) and then is induced to higher levels during lytic reactivation; however, PAN RNA is either the most abundant or among the most abundant transcripts during de novo infection (35,36). PAN RNA has been reported to inhibit the expression of immune response genes such as interleukin-4 and gamma interferon, to interact with several viral and cellular proteins, to act as a global regulator of viral genes by interacting with cellular transcriptional regulators and chromatin-modifying proteins, and to potentially encode small peptides from noncanonical open reading frames (ORFs) (13,(26)(27)(28)(29)(37)(38)(39).…”

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confidence: 99%

Expression of the Antisense-to-Latency Transcript Long Noncoding RNA in Kaposi's Sarcoma-Associated Herpesvirus

Schifano

Corcoran

Kelkar

et al. 2017

The regulation of latency is central to herpesvirus biology. Recent transcriptome-wide surveys have uncovered evidence for promiscuous transcription across the entirety of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome and postulated the existence of multiple viral long noncoding RNAs (lncRNAs). Nextgeneration sequencing studies are highly dependent on the specific experimental approach and particular algorithms of analysis and therefore benefit from independent confirmation of the results. The antisense-to-latency transcript (ALT) lncRNA was discovered by genome-tiling microarray (Chandriani et al., J Virol 86:7934 -7942, 2010, https://doi.org/10.1128/JVI.00645-10). To characterize ALT in detail, we physically isolated this lncRNA by a strand-specific hybrid capture assay and then employed transcriptome sequencing and novel reverse transcription-PCR (RT-PCR) assays to distinguish all RNA species in the KSHV latency region. These methods confirm that ALT initiates at positions 120739/121012 and encodes a single splice site, which is shared with the 3=-coterminal K14-vGPCR/ORF74 mRNA, terminating at 130873 (GenBank accession number GQ994935), resulting in an ϳ10,000-nucleotide transcript. No shorter ALT isoforms were identified. This study also identified a novel intron within the LANA 5= untranslated region using a splice acceptor at 127888. In summary, ALT joins PAN/nut1/T1.1 as a bona fide lncRNA of KSHV with potentially important roles in viral gene regulation and pathogenesis.IMPORTANCE Increasing data support the importance of noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and lncRNAs, which have been shown to exert critical regulatory functions without coding for recognizable proteins. Defining the sequences of these ncRNAs is essential for future studies aiming to functionally characterize a specific ncRNA. Most lncRNA studies are highly dependent on high-throughput sequencing and bioinformatic analyses, few studies follow up on the initial predictions, and analyses are at times discordant. The manuscript characterizes one key viral lncRNA, ALT, by physically isolating ALT and by a sequencing-independent assay. It provides for a simple assay to monitor lncRNA expression in experimental and clinical samples. ALT is expressed antisense to the major viral latency transcripts encoding LANA as well as the viral miRNAs and thus has the potential to regulate this key part of the viral life cycle.

“…This suggests the existence of an additional, proteasome-independent mechanism for degradation of SP100 and PML. These observations indicate that in cells that support efficient lytic replication, in addition to virion-associated proteins, de novo-synthesized gene products, perhaps from copackaged viral mRNA delivered with the viral particle (25) or from very early transcripts, can contribute to the degradation of SP100 and, in particular, of PML. An attractive hypothesis would be that early gene products of RRV contribute to degradation of SP100 and PML and induce their nonproteasomal degradation, in addition to the effects of the tegument protein ORF75.…”

Section: Discussionmentioning

confidence: 99%

Viral FGARAT Homolog ORF75 of Rhesus Monkey Rhadinovirus Effects Proteasomal Degradation of the ND10 Components SP100 and PML

Hahn

Großkopf

Jungnickl

et al. 2016

Nuclear domain 10 (ND10) components restrict herpesviral infection, and herpesviruses antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. The rhesus monkey rhadinovirus (RRV) shares many key biological features with the closely related Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) and readily infects cells of both human and rhesus monkey origin. We used the clustered regularly interspaced short palindromic repeatCas9 (CRISPR-Cas9) technique to generate knockout (ko) cells for each of the four ND10 components, PML, SP100, DAXX, and ATRX. These ko cells were analyzed with regard to permissiveness for RRV infection. In addition, we analyzed the fate of the individual ND10 components in infected cells by immunofluorescence and Western blotting. Knockout of the ND10 component DAXX markedly increased RRV infection, while knockout of PML or SP100 had a less pronounced effect. In line with these observations, RRV infection resulted in rapid degradation of SP100, followed by degradation of PML and the loss of ND10 structures, whereas the protein levels of ATRX and DAXX remained constant. Notably, inhibition of the proteasome but not inhibition of de novo gene expression prevented the loss of SP100 and PML in cells that did not support lytic replication, compatible with proteasomal degradation of these ND10 components through the action of a viral tegument protein. Expression of the RRV FGARAT homolog ORF75 was sufficient to effect the loss of SP100 and PML in transfected or transduced cells, implicating ORF75 as the viral effector protein. IMPORTANCEOur findings highlight the antiviral role of ND10 and its individual components and further establish the viral FGARAT homologs of the gammaherpesviruses to be important viral effectors that counteract ND10-instituted intrinsic immunity. Surprisingly, even closely related viruses like KSHV and RRV evolved to use different strategies to evade ND10-mediated restriction. RRV first targets SP100 for degradation and then targets PML with a delayed kinetic, a strategy which clearly differs from that of other gammaherpesviruses. Despite efficient degradation of these two major ND10 components, RRV is still restricted by DAXX, another abundant ND10 component, as evidenced by a marked increase in RRV infection and replication upon knockout of DAXX. Taken together, our findings substantiate PML, SP100, and DAXX as key antiviral proteins, in that the first two are targeted for degradation by RRV and the last one still potently restricts replication of RRV. T he rhesus monkey rhadinovirus (RRV) is a gamma-2-herpesvirus (rhadinovirus) naturally occurring in rhesus macaques (Macaca mulatta) and is closely related to the only human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV) (1). While RRV is usually apathogenic, at least isolate 17577 of RRV has been demonstrated to induce lymphomas and mesenchymal tumors after experimental RRV infection in the context of simian immunodeficiency vir...