Kaposi's Sarcoma-associated Herpesvirus Latency Research Articles

Nucleolin Regulates the Expression of Kaposi's Sarcoma-Associated Herpesvirus' Latency-Associated Nuclear Antigen through G-Quadruplexes in the mRNA.

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.

Distinctive Kaposi Sarcoma-Associated Herpesvirus Serological Profile during Acute Plasmodium falciparum Malaria Episodes

The seroprevalence of Kaposi sarcoma-associated herpesvirus (KSHV) and the incidence of endemic Kaposi sarcoma (KS) overlap with regions of malaria endemicity in sub-Saharan Africa. Multiple studies have shown an increased risk of KSHV seroconversion in children from high malaria compared to low malaria regions; however, the impact of acute episodes of Plasmodium falciparum (P. falciparum) malaria on KSHV's biphasic life cycle and lytic reactivation has not been determined. Here, we examined KSHV serological profiles and viral loads in 134 children with acute malaria and 221 healthy children from high malaria regions in Kisumu, as well as 77 healthy children from low malaria regions in Nandi. We assayed KSHV, Epstein-Barr virus (EBV), and P. falciparum malaria antibody responses in these three by multiplexed Luminex assay. We confirmed that KSHV seroprevalence was significantly associated with malaria endemicity (OR = 1.95, 1.18-3.24 95% CI, p = 0.01) with 71-77% seropositivity in high-malaria (Kisumu) compared to 28% in low-malaria (Nandi) regions. Furthermore, KSHV serological profiles during acute malaria episodes were distinct from age-matched non-malaria-infected children from the same region. Paired IgG levels also varied after malaria treatment, with significantly higher anti-ORF59 at day 0 but elevated ORF38, ORF73, and K8.1 at day 3. Acute malaria episodes is characterized by perturbation of KSHV latency in seropositive children, providing further evidence that malaria endemicity contributes to the observed increase in endemic KS incidence in sub-Saharan Africa.

KSHV hijacks FoxO1 to promote cell proliferation and cellular transformation by antagonizing oxidative stress.

Reactive oxygen species (ROS) are a group of a highly short-lived molecules that control diverse behaviors of cells. Normal cells maintain ROS balance to ensure their functions. Because of oncogenic stress, cancer cells often have excessive ROS, also known as oxidative stress, which are often counteracted by enhanced antioxidant systems to maintain redox homeostasis. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with Kaposi's sarcoma (KS), which manifests hyper inflammation and oxidative stress as the hallmarks. We have previously shown that excessive ROS can disrupt KSHV latency by inducing viral lytic replication, leading to cell death. Paradoxically, most KS tumor cells are latently infected by KSHV in a highly inflammatory and oxidative stress tumor microenvironment, which is in part due to the activation of alternative complement and TLR4 pathways, indicating the existence of an enhanced antioxidant defense system in KS tumor cells. In this study, we show that KSHV upregulates antioxidant genes, including SOD2 and CAT by hijacking the forkhead box protein O1 (FoxO1), to maintain intracellular ROS level. Moreover, the fine-tuned balance of ROS level in KSHV-transformed cells is essential for cell survival. Consequently, KSHV-transformed cells are extremely sensitive to exogenous ROS insult such as treatment with a low level of hydrogen peroxide (H2 O2 ). Either chemical inhibition or knockdown of FoxO1 by short interfering RNAs decreases the expression of antioxidant genes and subsequently increases the intracellular ROS level in KSHV-transformed cells, resulting in the inhibition of cell proliferation and colony formation in soft agar. Mechanistically, KSHV-encoded microRNAs and vFLIP upregulate FoxO1 by activating the NF-κB pathway. These results reveal a novel mechanism by which an oncogenic virus counteracts oxidative stress by upregulating FoxO1, which is essential for KSHV-induced cell proliferation and cellular transformation. Therefore, FoxO1 might be a potential therapeutic target for KSHV-related malignancies.

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposi's sarcoma (KS). We previously demonstrated that the KSHV Kaposin B (KapB) protein promotes inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). PBs modify gene expression by silencing or degrading labile messenger RNAs (mRNAs), including many transcripts that encode inflammatory or angiogenic proteins associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via phosphorylation of the autophagy regulatory protein, Beclin. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin phosphorylation or autophagic flux. Moreover, we showed that PB disassembly mediated by KSHV or KapB, depended on autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.

Nm23-H1 induces apoptosis in primary effusion lymphoma cells via inhibition of NF-κB signaling through interaction with oncogenic latent protein vFLIP K13 of Kaposi's sarcoma-associated herpes virus.

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin lymphoma of B cells caused by Kaposi's Sarcoma-associated Herpes Virus (KSHV). KSHV encoded latent and lytic antigens promote oncogenic transformation and evade apoptosis through the modulation of various host cellular signaling pathways. Nm23-H1 is a known metastatic suppressor whose expression inversely correlates with the metastatic potential of different cancers. Here, we set out to assess the role of Nm23-H1 in PEL development. Flow cytometry and real-time PCR assays were performed for Nm23-H1 expression analysis. Induction of apoptosis was assessed using Western blotting and flow cytometry-based assays in Nm23-H1 overexpressing cells. Co-immunoprecipitation assays, confocal microscopy and imaging flow cytometry were performed to determine Nm23-H1 and vFLIP K13 protein-protein interaction. A PEL cell-induced xenograft model was established in non-obese diabetic/severely combined immunodeficient (NOD/SCID) mice to validate the effect of Nm23-H1 overexpression. We found that Nm23-H1 expression was significantly downregulated both at transcriptional and protein levels in PEL cell lines and that its overexpression triggered mitochondrial-mediated caspase-dependent apoptosis. We revealed Nm23-H1 interacts with the latent protein vFLIP K13 and that Nm23-H1 overexpression leads to inhibition of vFLIP K13 driven nuclear factor-kappa B (NF-κB) signaling with concurrent inhibition of autocrine and paracrine growth factors and downregulation of latent KSHV antigens without induction of active lytic reactivation. We also confirmed the effects of Nm23-H1 overexpression in a PEL cell-induced xenograft model in NOD/SCID mice. Downregulation of Nm23-H1 expression in KSHV-infected PEL cells and its overexpression trigger apoptosis by impairing vFLIP K13-driven NF-κB signaling, suggesting therapeutic implications of Nm23-H1 for primary effusion lymphomas.

VFLIP SIM Promotes Viral Latency of KSHV

Kaposi’s Sarcoma‐Associated Herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), Primary Effusion Lymphoma (PEL), and Multicentric Castleman’s Disease (MCD). Upon primary infection, the KSHV genome primarily exists as a latent infection, tethered to the host chromatin as an episome. It is widely accepted that NFκB signaling promotes latency and that NFκB signaling is activated through expression of the viral FLICE Inhibitory Protein (vFLIP). Our objective is to reveal new insights into the regulation of KSHV latency. We have identified a putative SUMO Interaction Motif (SIM) in vFLIP and we hypothesize that the vFLIP SIM is required for maintaining latency. Mutation of this SIM abolishes the ability of vFLIP to activate NFκB signaling. We also observed reduced vFLIP degradation by the master lytic switch RTA, as well as increased lytic reactivation. Our data present a novel means by which KSHV vFLIP functions to maintain latency.

VFLIP-regulated competing endogenous RNA (ceRNA) networks targeting lytic induction for KSHV-associated malignancies.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes life-long latent infection and malignancies, including KScommonly found in AIDS patients. Lytic replication can be induced to kill tumor cells harboring latent KSHV, through viral cytopathic effects and the subsequent antiviral immune responses. Viral FLICE-inhibitory protein (vFLIP), encoded by KSHV ORF K13, inhibits KSHV lytic reactivation, implying that the competing endogenous RNA (ceRNA) networks regulated by vFLIP can be modulated to induce the lytic reactivation of latent KSHV, a promising strategy for KSHV-associated malignancies. Here, we performed whole-transcriptome sequencing to reveal the global landscape of noncoding RNAs and messenger RNAs (mRNAs) in iSLK-RGB-BAC16 cells and iSLK-RGB-K13 mutant cells. It showed that vFLIP regulated 227 differentially expressed (DE) long non-coding RNAs (lncRNAs), 57 DEcircular RNAs(circRNAs), 20 DE microRNAs (miRNAs), and 1371 DE mRNAs. Enrichment analysis verified that riboflavin metabolism was simultaneously enriched in DE genes related to miRNAs, lncRNAs, and circRNAs. The upregulated hsa-miR-378i and hsa-miR-3654, and downregulated miR-4467, miR-3163, miR-4451, and miR-4257 were significantly enriched in the ceRNA complex network, which contained 9 upregulated and 7 downregulated circRNAs, 5 upregulated and 85 downregulated lncRNAs, 5 upregulated and 35 downregulated mRNAs. Finally, we constructed and validated two vFLIP-regulated ceRNA networks: circRNA hsa_circ_0070049/hsa-miR-378i/SPEG/FOXQ1 and lncRNA AL031123.1/hsa-miR-378i/SPEG/FOXQ1. Taken together, the two ceRNA networks may mediate KSHV reactivation. These novel findings refreshed the present understanding of ceRNA network in KSHV lytic induction and provided potential therapeutic targets for KSHV-associated malignancies.

A Panel of Kaposi's Sarcoma-Associated Herpesvirus Mutants in the Polycistronic Kaposin Locus for Precise Analysis of Individual Protein Products.

ABSTRACTKaposi’s sarcoma-associated herpesvirus (KSHV) is the cause of several human cancers, including the endothelial cell (EC) malignancy, Kaposi’s sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, the “K-genes,” are important for KSHV replication and pathogenesis. Among these, the kaposin transcript is highly expressed in all phases of infection, but its complex polycistronic nature has hindered functional analysis to date. At least three proteins are produced from the kaposin transcript: Kaposin A (KapA), B (KapB), and C (KapC). To determine the relative contributions of kaposin proteins during KSHV infection, we created a collection of mutant viruses unable to produce kaposin proteins individually or in combination. In previous work, we showed KapB alone recapitulated the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). Using the new ΔKapB virus, we showed that KapB was necessary for this effect during latent KSHV infection. Moreover, we observed that despite the ability of all kaposin-deficient latent iSLK cell lines to produce virions, all displayed low viral episome copy number, a defect that became more pronounced after primary infection of naive ECs. For ΔKapB, provision of KapB in trans failed to complement the defect, suggesting a requirement for the kaposin locus in cis. These findings demonstrate that our panel of kaposin-deficient viruses enables precise analysis of the respective contributions of individual kaposin proteins to KSHV replication. Moreover, our mutagenesis approach serves as a guide for the functional analysis of other complex multicistronic viral loci.IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) expresses high levels of the kaposin transcript during both latent and lytic phases of replication. Due to its repetitive, GC-rich nature and polycistronic coding capacity, until now no reagents existed to permit a methodical analysis of the role of individual kaposin proteins in KSHV replication. We report the creation of a panel of recombinant viruses and matched producer cell lines that delete kaposin proteins individually or in combination. We demonstrate the utility of this panel by confirming the requirement of one kaposin translation product to a key KSHV latency phenotype. This study describes a new panel of molecular tools for the KSHV field to enable precise analysis of the roles of individual kaposin proteins during KSHV infection.

MLL1 is regulated by KSHV LANA and is important for virus latency.

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-Å crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.

Narciclasine Display Preferential Pre-Clinical Activity Against KSHV-Associated Primary Effusion Lymphoma By Targeting MYC Via NF-κB

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma with extremely poor prognosis when treated with conventional chemotherapy. Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of PEL. KSHV-encoded viral FLICE-inhibitory protein (vFLIP)-K13 constitutively activates the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. In a K13-isogenic cell line based screening of small molecule libraries, we have identified the natural products narciclasine and its structural analog, 7-deoxynarciclasine (found in daffodils), as having preferential cytotoxicity towards K13-expressing isogenic cell line. Further, the structurally closely related lycorine (another alkaloid found in daffodils) also exhibited similar activity. All the three compounds (Narciclasine, 7-deoxynarciclasine, and lycorine) display preferential cytotoxicity against PEL in a panel of cell lines comprising 6 hematological malignancies. Narciclasine is approximately 10 and 100-fold much effective than its structural analogs, 7-deoxynarciclasine, and lycorine, respectively. Importantly, IC50 of narciclasine in PEL cell lines (⁓10 nM) are well below the physiological concentration that was achieved in murine pharmacokinetic studies (250 nM). Narciclasine arrested cell cycle progression of PEL cells at the G0/G1 phase, with a concomitant decrease in the percentage of cells in S-phase. Further, there is a significant increase in the number of cells in sub-G0 peak, representative of apoptotic cells, which was confirmed by activation of caspase-3/7, AnnexinV staining, and PARP cleavage. Narciclasine blocked the NF-κB promoter activity in PEL cells as observed by a drastic decrease in firefly luciferase activity of PEL cells stably transduced with a lentiviral NF-κB-Luc reporter. IL-6 and IL-10 are known NF-κB targets and growth factors that are constitutively secreted by PEL, striking decrease in their secretory levels were observed upon treatment with narciclasine. Narciclasine preferentially blocked K13-induced NF-κB over the constitutive NF-κB activity in cells, which were engineered to express K13 conditionally (tet-on inducible lentiviral system). IKKγ/NEMO is essential for K13-induced NF-κB. The preference of narciclasine for K13-induced NF-κB is completely abolished upon NEMO knockout by CRISPR-Cas9 system. While probing the effect of narciclasine on the levels of putative NF-κB target genes by qRT-PCR in PEL, we found that there is a significant decrease in mRNA level of MYC, with a concomitant decrease in its protein level. Subsequently, all the direct target genes of MYC (MYB, TYRO3, TERT, PMM2, SLC19A1, and HK2) were significantly down regulated upon treatment with narciclasine. Furthermore, ectopic expression of MYC from a retroviral promoter significantly diminished the anti-proliferative effect of narciclasine and its analogs in PEL. It has been shown that inhibition of MYC is toxic to PEL and compounds that target MYC display selective activity against PEL, which explains the preferential activity of narciclasine and its analogs in PEL. MYC is required for maintenance of KSHV latency, and its down regulation results in the induction of KSHV Replication and Transcription Activator (RTA/ORF50) in PEL. However, narciclasine neither induced the expression of RTA nor the production of infectious KSHV virions in PEL. Most importantly, narciclasine doubled the survival of NSG mice bearing orthotopic - Cell Line Xenograft (CLX) and Patient Derived Xenograft (PDX) models of PEL. Finally, the IC50 of narciclasine in PBMCs from normal healthy donors is approximately 4-fold higher than the PEL cells directly isolated from the mice bearing the PDX model of the disease and PEL cell lines, indicating that there is a significant therapeutic window for narciclasine, which can be exploited for the treatment of KSHV-associated malignancies. DisclosuresChaudhary:University of Southern California: Patents & Royalties: P.M.C. have filed patent application related to the technology described in this manuscript.

A CRISPR-Cas9 screen identifies mitochondrial translation as an essential process in latent KSHV infection of human endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The main proliferating component of KS tumors is a cell of endothelial origin termed the spindle cell. Spindle cells are predominantly latently infected with only a small percentage of cells undergoing viral replication. As there is no direct treatment for latent KSHV, identification of host vulnerabilities in latently infected endothelial cells could be exploited to inhibit KSHV-associated tumor cells. Using a pooled CRISPR-Cas9 lentivirus library, we identified host factors that are essential for the survival or proliferation of latently infected endothelial cells in culture, but not their uninfected counterparts. Among the many host genes identified, there was an enrichment in genes localizing to the mitochondria, including genes involved in mitochondrial translation. Antibiotics that inhibit bacterial and mitochondrial translation specifically inhibited the expansion of latently infected endothelial cells and led to increased cell death in patient-derived PEL cell lines. Direct inhibition of mitochondrial respiration or ablation of mitochondrial genomes leads to increased death in latently infected cells. KSHV latent infection decreases mitochondrial numbers, but there are increases in mitochondrial size, genome copy number, and transcript levels. We found that multiple gene products of the latent locus localize to the mitochondria. During latent infection, KSHV significantly alters mitochondrial biology, leading to enhanced sensitivity to inhibition of mitochondrial respiration, which provides a potential therapeutic avenue for KSHV-associated cancers.

Global epigenomic analysis of KSHV-infected primary effusion lymphoma identifies functional MYC superenhancers and enhancer RNAs.

Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.

Epigenetic Regulation of Kaposi's Sarcoma-Associated Herpesvirus Latency.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus that infects humans and exhibits a biphasic life cycle consisting of latent and lytic phases. Following entry into host cells, the KSHV genome undergoes circularization and chromatinization into an extrachromosomal episome ultimately leading to the establishment of latency. The KSHV episome is organized into distinct chromatin domains marked by variations in repressive or activating epigenetic modifications, including DNA methylation, histone methylation, and histone acetylation. Thus, the development of KSHV latency is believed to be governed by epigenetic regulation. In the past decade, interrogation of the KSHV epitome by genome-wide approaches has revealed a complex epigenetic mark landscape across KSHV genome and has uncovered the important regulatory roles of epigenetic modifications in governing the development of KSHV latency. Here, we highlight many of the findings regarding the role of DNA methylation, histone modification, post-translational modification (PTM) of chromatin remodeling proteins, the contribution of long non-coding RNAs (lncRNAs) in regulating KSHV latency development, and the role of higher-order episomal chromatin architecture in the maintenance of latency and the latent-to-lytic switch.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions.

Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infection-associated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection.IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment.

Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA.To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo.Using co-infection experiments and recombinant viruses, we also show that KSHV’s ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.

The landscape of transcription initiation across latent and lytic KSHV genomes.

Precise promoter annotation is required for understanding the mechanistic basis of transcription initiation. In the context of complex genomes, such as herpesviruses where there is extensive genic overlap, identification of transcription start sites (TSSs) is particularly problematic and cannot be comprehensively accessed by standard RNA sequencing approaches. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and the etiological agent of Kaposi’s sarcoma and the B cell lymphoma primary effusion lymphoma (PEL). Here, we leverage RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) and define KSHV TSSs transcriptome-wide and at nucleotide resolution in two widely used models of KSHV infection, namely iSLK.219 cells and the PEL cell line TREx-BCBL1-RTA. By mapping TSSs over a 96 h time course of reactivation we confirm 48 of 50 previously identified TSSs. Moreover, we identify over 100 novel transcription start site clusters (TSCs) in each cell line. Our analyses identified cell-type specific differences in TSC positions as well as promoter strength, and defined motifs within viral core promoters. Collectively, by defining TSSs at high resolution we have greatly expanded the transcriptional landscape of the KSHV genome and identified transcriptional control mechanisms at play during KSHV lytic reactivation.

Reduction of Kaposi's Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), an AIDS-defining cancer in HIV-1-infected individuals or immune-suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV's ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome but also Epstein-Barr virus (EBV) which, similar to KSHV, belongs to the Gammaherpesvirus family. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication, and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver a LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of an adenovirus vector might confer potential in vivo applications of LANA-specific Cas9 against KSHV infection and KS.IMPORTANCE The ability for Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potential benefits in developing countries where the viral cancer burden is high.

Latent infection with Kaposi's sarcoma-associated herpesvirus enhances retrotransposition of long interspersed element-1.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a gamma-2 herpesvirus, is the causative agent of KS, primary effusion lymphoma (PEL), and a plasma cell variant of multicentric Castleman's disease. Although KSHV latency is detected in KS-related tumors, oncogenic pathways activated by KSHV latent infection are not fully understood. Here, we found that retrotransposition of long interspersed element-1 (L1), a retrotransposon in the human genome, was enhanced in PEL cells. Among the KSHV latent genes, viral FLICE-inhibitory protein (vFLIP) enhanced L1 retrotransposition in an NF-κB-dependent manner. Intracellular cell adhesion molecule-1 (ICAM-1), an NF-κB target, regulated the vFLIP-mediated enhancement of L1 retrotransposition. Furthermore, ICAM-1 downregulated the expression of Moloney leukemia virus 10 (MOV10), an L1 restriction factor. Knockdown of ICAM-1 or overexpression of MOV10 relieved the vFLIP-mediated enhancement of L1 retrotransposition. Collectively, during KSHV latency, vFLIP upregulates ICAM-1 in an NF-κB-dependent manner, which, in turn, downregulates MOV10 expression and thereby enhances L1 retrotransposition. Because active L1 retrotransposition can lead to genomic instability, which is commonly found in KS and PEL, activation of L1 retrotransposition during KSHV latency may accelerate oncogenic processes through enhancing genomic instability. Our results suggest that L1 retrotransposition may be a novel target for impeding tumor development in KSHV-infected patients.

Mast Cell Activation and KSHV Infection in Kaposi Sarcoma.

Purpose: Kaposi sarcoma (KS) is a vascular tumor initiated by infection of endothelial cells (ECs) with KS-associated herpesvirus (KSHV). KS is dependent on sustained proinflammatory signals provided by intralesional leukocytes and continued infection of new ECs. However, the sources of these cytokines and infectious virus within lesions are not fully understood. Here, mast cells (MCs) are identified as proinflammatory cells within KS lesions that are permissive for, and activated by, infection with KSHV.Experimental Design: Three validated MC lines were used to assess permissivity of MCs to infection with KSHV and to evaluate MCs activation following infection. Biopsies from 31 AIDS-KS cases and 11 AIDS controls were evaluated by IHC for the presence of MCs in KS lesions and assessment of MC activation state and infection with KSHV. Plasma samples from 26 AIDS-KS, 13 classic KS, and 13 healthy adults were evaluated for levels of MC granule contents tryptase and histamine.Results: In culture, MCs supported latent and lytic KSHV infection, and infection-induced MC degranulation. Within KS lesions, MCs were closely associated with spindle cells. Furthermore, MC activation was extensive within patients with KS, reflected by elevated circulating levels of tryptase and a histamine metabolite. One patient with clinical signs of extensive MC activation was treated with antagonists of MC proinflammatory mediators, which resulted in a rapid and durable regression of AIDS-KS lesions.Conclusions: Using complimentary in vitro and in vivo studies we identify MCs as a potential long-lived reservoir for KSHV and a source of proinflammatory mediators within the KS lesional microenvironment. In addition, we identify MC antagonists as a promising novel therapeutic approach for KS. Clin Cancer Res; 24(20); 5085-97. ©2018 AACR.

Kaposi's Sarcoma-Associated Herpesvirus Latency Locus Renders B Cells Hyperresponsive to Secondary Infections.

Kaposi's sarcoma-associated herpesvirus (KSHV) induces B cell hyperplasia and neoplasia, such as multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL). To explore KSHV-induced B cell reprogramming in vivo, we expressed the KSHV latency locus, inclusive of all viral microRNAs (miRNAs), in B cells of transgenic mice in the absence of the inhibitory FcγRIIB receptor. The BALB/c strain was chosen as this is the preferred model to study B cell differentiation. The mice developed hyperglobulinemia, plasmacytosis, and B lymphoid hyperplasia. This phenotype was ameliorated by everolimus, which is a rapamycin derivative used for the treatment of mantle cell lymphoma. KSHV latency mice exhibited hyperresponsiveness to the T-dependent (TD) antigen mimic anti-CD40 and increased incidence of pristane-induced inflammation. Lastly, the adaptive immunity against a secondary infection with Zika virus (ZIKV) was markedly enhanced. These phenotypes are consistent with KSHV lowering the activation threshold of latently infected B cells, which may be beneficial in areas of endemicity, where KSHV is acquired in childhood and infections are common.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latency in B cells and is stringently linked to primary effusion lymphoma (PEL) and the premalignant B cell hyperplasia multicentric Castleman's disease (MCD). To investigate potential genetic background effects, we expressed the KSHV miRNAs in BALB/c transgenic mice. BALB/c mice are the preferred strain for B cell hybridoma development because of their propensity to develop predictable B cell responses to antigen. The BALB/c latency mice exhibited a higher incidence of B cell hyperplasia as well as sustained hyperglobulinemia. The development of neutralizing antibodies against ZIKV was augmented in BALB/c latency mice. Hyperglobulinemia was dampened by everolimus, a derivative of rapamycin, suggesting a role for mTOR inhibitors in managing immune activation, which is hallmark of KSHV infection as well as HIV infection.