Expression Ratios of the Antiapoptotic BCL2 Family Members Dictate the Selective Addiction of Kaposi's Sarcoma-Associated Herpesvirus-Transformed Primary Effusion Lymphoma Cell Lines to MCL1.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people living with HIV, including primary effusion lymphoma (PEL). PEL cell lines exhibit oncogene addictions to both viral and cellular genes. Using CRISPR screens, we previously identified cellular oncogene addictions in PEL cell lines, including MCL1. MCL1 is a member of the BCL2 family, which functions to prevent intrinsic apoptosis and has been implicated in several cancers. Despite the overlapping functions of the BCL2 family members, PEL cells are dependent only on MCL1, suggesting that MCL1 may have nonredundant functions. To investigate why PEL cells exhibit selective addiction to MCL1, we inactivated the intrinsic apoptosis pathway by engineering BAX/BAK1 double knockout cells. In this context, PEL cells become resistant to MCL1 knockdown or MCL1 inactivation by the MCL1 inhibitor S63845, indicating that the main function of MCL1 in PEL cells is to prevent BAX/BAK1-mediated apoptosis. The selective requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Ectopic expression of several BCL2 family proteins, as well as the KSHV BCL2 homolog, significantly decreased basal caspase 3/7 activity and buffered against staurosporine-induced apoptosis. Finally, overexpressed BCL2 family members can functionally substitute for MCL1, when it is inhibited by S63845. Together, our data indicate that the expression levels of the BCL2 family likely explain why PEL tumor cells are highly addicted to MCL1. Importantly, our results suggest that caution should be taken when considering MCL1 inhibitors as a monotherapy regimen for PEL because resistance can develop easily. IMPORTANCE Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus. We showed previously that PEL cell lines require the antiapoptotic protein MCL1 for survival but not the other BCL2 family proteins. This selective dependence on MCL1 is unexpected as the BCL2 family functions similarly in preventing intrinsic apoptosis. Recently, new roles for MCL1 not shared with the BCL2 family have emerged. Here, we show that noncanonical functions of MCL1 are unlikely essential. Instead, MCL1 functions mainly to prevent apoptosis. The specific requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Consistent with this model, shifting these expression ratios changes the requirement away from MCL1 and toward the dominant BCL2 family gene. Together, our results indicate that although MCL1 is an attractive chemotherapeutic target to treat PEL, careful consideration must be taken, as resistance to MCL1-specific inhibitors easily develops through BCL2 family overexpression.

DNA damage triggers an interplay between wtp53 and c-Myc affecting lymphoma cell proliferation and Kaposi sarcoma herpesvirus replication.

The induction of DNA damage together with the interference with DNA repair represents a promising strategy in cancer treatment. Here we show that the PARP-1/2/3 inhibitor AZD2461 in combination with the CHK1 inhibitor UCN-01 altered the DNA damage response and reduced cell proliferation in PEL cells, an aggressive B cell lymphoma highly resistant to chemotherapies. AZD2461/UCN-01 combination activated p53/p21 and downregulated c-Myc in these cells, leading to a reduced expression level of RAD51, molecule involved in DNA repair. The effect of AZD2461/UCN-01 on c-Myc and p53/p21 was inter-dependent and, besides impairing cell proliferation, contributed to the activation of the replicative cycle of KSHV, carried in a latent state in PEL cells. Finally, we found that the pharmacological or genetic inhibition of p21 counteracted the viral lytic cycle activation and further reduced PEL cell proliferation, suggesting that it could induce a double beneficial effect in this setting. This study unveils that, therapeutic approaches, based on the induction of DNA damage and the reduction of DNA repair, could be used to successfully treat this malignant lymphoma.

A Comprehensive Clinicopathologic and Molecular Study of 19 Primary Effusion Lymphomas in HIV-infected Patients.

Primary effusion lymphoma (PEL) is associated with human herpesvirus 8 and frequently with Epstein-Barr virus (EBV). We report here a single-center series of 19 human immunodeficiency virus-associated PELs, including 14 EBV+ and 5 EBV- PELs. The objectives were to describe the clinicopathologic features of PELs, with a focus on programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) expression, to search for genetic alterations by targeted deep sequencing analysis, and to compare the features between EBV+ and EBV- cases. All the patients were male, and the median age at diagnosis was 47 years old (interquartile range: 40 to 56 y). Reflecting the terminal B-cell differentiation, immunophenotypic profiles showed low expression levels of B-cell markers, including CD19 (0/19), CD20 (1/19), CD79a (0/19), PAX5 (1/19), BOB1 (3/19), and OCT2 (4/19), contrasting with a common expression of CD38 (10/19), CD138 (7/19), and IRF4/MUM1 (18/19). We observed a frequent aberrant expression of T-cell markers, especially CD3 (10/19), and less frequently CD2 (2/19), CD4 (3/19), CD5 (1/19), and CD8 (0/19). Only 2 cases were PD-L1 positive on tumor cells and none PD-1 positive. With respect to immune cells, 3 samples tested positive for PD-L1 and 5 for PD-1. Our 36-gene lymphopanel revealed 7 distinct variants in 5/10 PELs, with either a single or 2 mutations per sample: B2M (n=2), CD58 (n=1), EP300 (n=1), TNFAIP3 (n=1), ARID1A (n=1), and TP53 (n=1). Finally, we did not observe any major clinical, pathologic, or immunohistochemical differences between EBV+ and EBV- PELs and the outcome was similar (2-y overall survival probability of 61.9% [95% confidence interval, 31.2-82.1] vs. 60.0% [95% confidence interval, 12.6-88.2], respectively, P=0.62).

The ORF45 Protein of Kaposi Sarcoma-Associated Herpesvirus Is an Inhibitor of p53 Signaling during Viral Reactivation.

Kaposi sarcoma-associated herpesvirus (KSHV) is a carcinogenic double-stranded DNA virus and the etiological agent of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). To prevent premature apoptosis and support its replication cycle, KSHV expresses a series of open reading frames (ORFs) that regulate signaling by the p53 tumor suppressor protein. Here, we describe a novel viral inhibitor of p53 encoded by KSHV ORF45 and identify its mechanism of action. ORF45 binds to p53 and prevents its interactions with USP7, a p53 deubiquitinase. This results in decreased p53 accumulation, localization of p53 to the cytoplasm, and diminished transcriptional activity. IMPORTANCE Unlike in other cancers, the tumor suppressor protein p53 is rarely mutated in Kaposi sarcoma (KS). Rather, Kaposi sarcoma-associated herpesvirus (KSHV) inactivates p53 through multiple viral proteins. One possible therapeutic approach to KS is the activation of p53, which would result in apoptosis and tumor regression. In this regard, it is important to understand all the mechanisms used by KSHV to modulate p53 signaling. This work describes a novel inhibitor of p53 signaling and a potential drug target, ORF45, and identifies the mechanisms of its action.

How Epstein-Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Are Maintained Together to Transform the Same B-Cell.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) independently cause human cancers, and both are maintained as plasmids in tumor cells. They differ, however, in their mechanisms of segregation; EBV partitions its genomes quasi-faithfully, while KSHV often clusters its genomes and partitions them randomly. Both viruses can infect the same B-cell to transform it in vitro and to cause primary effusion lymphomas (PELs) in vivo. We have developed simulations based on our measurements of these replicons in B-cells transformed in vitro to elucidate the synthesis and partitioning of these two viral genomes when in the same cell. These simulations successfully capture the biology of EBV and KSHV in PELs. They have revealed that EBV and KSHV replicate and partition independently, that they both contribute selective advantages to their host cell, and that KSHV pays a penalty to cluster its genomes.

[Human immunodeficiency virus and lymphoma]. / Virus de l'immunodéficience humaine et lymphome.

Lymphomas remain a leading cause of morbidity and mortality for HIV-positive patients. The most common lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma, primary effusion lymphoma, plasmablastic lymphoma and Hodgkin lymphoma. Appropriate approach is determined by lymphoma stage, performans status, comorbidities, histological subtype, status of the HIV disease and immunosuppression. Treatment outcomes have improved due to chemotherapy modalities and effective antiretroviral therapy. This review summarizes epidemiology, pathogenesis, pathology, and current treatment landscape in HIV associated lymphoma.

Primary effusion lymphoma occurring in the setting of transplanted patients: a systematic review of a rare, life-threatening post-transplantation occurrence.

BACKGROUND: Primary effusion lymphoma is a rare, aggressive large B-cell lymphoma strictly linked to infection by Human Herpes virus 8/Kaposi sarcoma-associated herpes virus. In its classic form, it is characterized by body cavities neoplastic effusions without detectable tumor masses. It often occurs in immunocompromised patients, such as HIV-positive individuals. Primary effusion lymphoma may affect HIV-negative elderly patients from Human Herpes virus 8 endemic regions. So far, rare cases have been reported in transplanted patients. The purpose of our systematic review is to improve our understanding of this type of aggressive lymphoma in the setting of transplantation, focusing on epidemiology, clinical presentation, pathological features, differential diagnosis, treatment and outcome. The role of assessing the viral serological status in donors and recipients is also discussed. METHODS: We performed a systematic review adhering to the PRISMA guidelines. The literature search was conducted on PubMed/MEDLINE, Web of Science, Scopus, EMBASE and Cochrane Library, using the search terms "primary effusion lymphoma" and "post-transplant". RESULTS: Our search identified 13 cases of post-transplant primary effusion lymphoma, predominantly in solid organ transplant recipients (6 kidney, 3 heart, 2 liver and 1 intestine), with only one case after allogenic bone marrow transplantation. Long-term immunosuppression is important in post-transplant primary effusion lymphoma commonly developing several years after transplantation. Kaposi Sarcoma occurred in association with lymphoma in 4 cases of solid organ recipients. The lymphoma showed the classical presentation with body cavity effusions in absence of tumor masses in 10 cases; 2 cases presented as solid masses, lacking effusions and one case as effusions associated with multiple organ involvement. Primary effusion lymphoma occurring in the setting of transplantation was more often Epstein Barr-virus negative. The prognosis was poor. In addition to chemotherapy, reduction of immunosuppressive treatment, was generally attempted. CONCLUSIONS: Primary effusion lymphoma is a rare, but often fatal post-transplant complication. Its rarity and the difficulty in achieving the diagnosis may lead to miss this complication. Clinicians should suspect primary effusion lymphoma in transplanted patients, presenting generally with unexplained body cavity effusions, although rare cases with solid masses are described.

Cucurbitacin B induces apoptosis of primary effusion lymphoma via disruption of cytoskeletal organization.

BACKGROUND: Primary effusion lymphoma (PEL) is an aggressive B cell non-Hodgkin lymphoma that develops especially in AIDS patients and immunocompromised patients infected with human herpes virus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus (KSHV). PEL has a poor prognosis in patients despite conventional chemotherapeutic treatment, and a safe and efficient therapy is required. PURPOSE: To examine the effects on PEL of cucurbitacin B (CuB), a triterpene found in plants of the Cucurbitaceae family that has several anti-cancer activities. STUDY DESIGN: We evaluated the anti-cancer activities of CuB in vitro and in vivo. METHODS: Cell proliferation of PEL cell lines was measured by MTT assay. Cleaved caspases and signaling transduction associated proteins were analyzed by western blotting. Wright and Giemsa staining and immunofluorescence staining were carried out to observe cell morphology. Cell cycles were analyzed by flow cytometry. RT-PCR was performed to detect viral gene expressions. A xenograft mouse model was employed to evaluate the anti-cancer activity of CuB in vivo. RESULTS: CuB inhibited cell proliferation of PEL cell lines (BCBL-1, BC-1, GTO and TY-1) in a dose-dependent manner (0-50 nM) and induced apoptosis of BCBL-1 cells via caspase activation in a dose- and time-dependent manner. In addition, CuB caused cell-shape disruption by inducing actin aggregation and suppressing the p-cofilin level, resulting in BCBL-1 cell arrest at the G2/M phase. In contrast, CuB showed almost no suppression of p-STAT3 and p-Akt activation, which were constitutively activated by KSHV-derived proteins. Furthermore, CuB (0.5 mg/kg) via intraperitoneal injection significantly (p < 0.05) suppressed solid tumor growth in the xenograft mouse model. CONCLUSION: This study suggests that CuB is a promising agent for PEL treatment.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen dysregulates expression of MCL-1 by targeting FBW7.

Primary effusion lymphoma (PEL) is an aggressive B cell lymphoma that is etiologically linked to Kaposi's sarcoma-associated herpesvirus (KSHV). Despite standard multi-chemotherapy treatment, PEL continues to cause high mortality. Thus, new strategies to control PEL are needed urgently. Here, we show that a phosphodegron motif within the KSHV protein, latency-associated nuclear antigen (LANA), specifically interacts with E3 ubiquitin ligase FBW7, thereby competitively inhibiting the binding of the anti-apoptotic protein MCL-1 to FBW7. Consequently, LANA-FBW7 interaction enhances the stability of MCL-1 by preventing its proteasome-mediated degradation, which inhibits caspase-3-mediated apoptosis in PEL cells. Importantly, MCL-1 inhibitors markedly suppress colony formation on soft agar and tumor growth of KSHV+PEL/BCBL-1 in a xenograft mouse model. These results strongly support the conclusion that high levels of MCL-1 expression enable the oncogenesis of PEL cells and thus, MCL-1 could be a potential drug target for KSHV-associated PEL. This work also unravels a mechanism by which an oncogenic virus perturbs a key component of the ubiquitination pathway to induce tumorigenesis.

Human herpesvirus-8-positive primary effusion lymphoma in HIV-negative patients: Single institution case series with a multidisciplinary characterization.

BACKGROUND: Primary effusion lymphoma (PEL) is a very rare non-Hodgkin lymphoma caused by human herpesvirus-8 (HHV8) that grows in liquid phase within body cavities. The diagnosis of PEL is based on cytology but requires confirmatory ancillary tests. PEL occurs mainly in association with HIV infection. This study describes 9 cases of PEL in HIV-negative patients and compares their characteristics with 10 HIV-associated cases of PEL diagnosed at a single institution in Italy between 1995 and 2019. METHODS: Clinical records were reviewed for demographic data, comorbidities, laboratory abnormalities, and outcome. PEL samples were evaluated for cytomorphology, immunophenotype, immunoglobulin (IG)/T cell receptor (TR) rearrangements, and HHV8 and Epstein-Barr virus (EBV) viral loads in effusion supernatants. RESULTS: HIV-unrelated PEL occurred in 8 elderly patients (7 men, 1 woman) and 1 young adult with primary antibody deficiency. Cytology revealed HHV8-positive lymphoma cells lacking B/T cell antigens and exhibiting 2 cell patterns (polymorphous or monotonous). IG was clonally rearranged in all cases; aberrant TRG occurred in 2 cases. Effusion supernatants had more than 106 HHV8 DNA copies per mL and variable loads of EBV DNA. Compared with HIV-associated PEL, the HIV-negative cohort was characterized by older age, less frequent association with Kaposi sarcoma and/or multicentric Castleman disease, comparable but less abnormal laboratory parameters, and a nonsignificant survival benefit. PEL cases with low apoptosis were associated with better prognosis. CONCLUSION: To the best of our knowledge, our case series of HIV-unrelated PEL is the largest thus far, expands the spectrum of cytological findings, and supports the need for a multidisciplinary approach in the diagnostic workup.

High levels of LINE-1 transposable elements expressed in Kaposi's sarcoma-associated herpesvirus-related primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is a gamma herpesvirus associated with several human malignancies. Transposable elements (TEs) are ubiquitous in eukaryotic genomes, occupying about 45% of the human genome. TEs have been linked with a variety of disorders and malignancies, though the precise nature of their contribution to many of them has yet to be elucidated. Global transcriptome analysis for differentially expressed TEs in KSHV-associated primary effusion lymphoma (PEL) cells (BCBL1 and BC3) revealed large number of differentially expressed TEs. These differentially expressed TEs include LTR transposons, long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs). Further analysis of LINE-1 (L1) elements revealed expression upregulation, hypo-methylation, and transition into open chromatin in PEL. In agreement with high L1 expression, PEL cells express ORF1 protein and possess high reverse transcriptase (RT)-activity. Interestingly, inhibition of this RT-activity suppressed PEL cell growth. Collectively, we identified high expression of TEs, and specifically of L1 as a critical component in the proliferation of PEL cells. This observation is relevant for the treatment of KSHV-associated malignancies since they often develop in AIDS patients that are treated with RT inhibitors with potent inhibition for both HIV and L1 RT activity.

Primary effusion lymphoma enhancer connectome links super-enhancers to dependency factors.

Primary effusion lymphoma (PEL) has a very poor prognosis. To evaluate the contributions of enhancers/promoters interactions to PEL cell growth and survival, here we produce H3K27ac HiChIP datasets in PEL cells. This allows us to generate the PEL enhancer connectome, which links enhancers and promoters in PEL genome-wide. We identify more than 8000 genomic interactions in each PEL cell line. By incorporating HiChIP data with H3K27ac ChIP-seq data, we identify interactions between enhancers/enhancers, enhancers/promoters, and promoters/promoters. HiChIP further links PEL super-enhancers to PEL dependency factors MYC, IRF4, MCL1, CCND2, MDM2, and CFLAR. CRISPR knock out of MEF2C and IRF4 significantly reduces MYC and IRF4 super-enhancer H3K27ac signal. Knock out also reduces MYC and IRF4 expression. CRISPRi perturbation of these super-enhancers by tethering transcription repressors to enhancers significantly reduces target gene expression and reduces PEL cell growth. These data provide insights into PEL molecular pathogenesis.

CRISPR/Cas9 ablating viral microRNA promotes lytic reactivation of Kaposi's sarcoma-associated herpesvirus.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system is an RNA-guided, DNA editing method that has been widely used for gene editing, including human viruses. Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), following latent infection in human cells, can cause a variety of malignancies, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), with a high prevalence in immunocompromised patients. Of significant concern, the latent infection with KSHV has been shown to lead to increased resistance to antiviral therapies. MicroRNAs (miRNAs) are a set of non-coding, small RNA molecules that regulate protein-coding genes at the post-transcriptional and translational levels. KSHV has its miRNAs, most of which are expressed in latently infected cells and play a crucial role in maintaining KSHV latency. Notably, by regulating the expression of the downstream target genes in host cells, KSHV miRNAs can interact with the host environment to promote the development of KSHV-related diseases. Although CRISPR/Cas9 has been reported to edit KSHV protein-coding genes, there is no published literature on whether the CRISPR/Cas9 system can regulate the expression of KSHV miRNAs. In this study, we used CRISPR/Cas9 to inhibit the expression of KSHV miRNAs by directly editing the DNA sequences of individual KSHV miRNAs, or the promoter of clustered KHSV miRNAs, in latent KSHV-infected PEL cells. Our results show that CRISPR/Cas9 can ablate KSHV miRNAs expression, which in turn leads to the upregulation of viral lytic genes and alteration of host cellular gene expression. To the best of our knowledge, our study is the first reported demonstration of the CRISPR/Cas9 system editing KSHV miRNAs, further expanding the application of CRISPR/Cas9 as a novel antiviral strategy targeting KSHV latency.

Regulation of KSHV Latency and Lytic Reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three malignancies- Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Central to the pathogenesis of these diseases is the KSHV viral life cycle, which is composed of a quiescent latent phase and a replicative lytic phase. While the establishment of latency enables persistent KSHV infection and evasion of the host immune system, lytic replication is essential for the dissemination of the virus between hosts and within the host itself. The transition between these phases, known as lytic reactivation, is controlled by a complex set of environmental, host, and viral factors. The effects of these various factors converge on the regulation of two KSHV proteins whose functions facilitate each phase of the viral life cycle-latency-associated nuclear antigen (LANA) and the master switch of KSHV reactivation, replication and transcription activator (RTA). This review presents the current understanding of how the transition between the phases of the KSHV life cycle is regulated, how the various phases contribute to KSHV pathogenesis, and how the viral life cycle can be exploited as a therapeutic target.

Kaposi's Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). The cellular transcription factor (TF) interferon (IFN) regulatory factor 4 (IRF4) is an essential oncogene in PEL, but its specific role in PEL and how KSHV deregulates IRF4 remain unknown. Here, we report that the KSHV latency protein viral interferon regulatory factor 3 (vIRF3) cooperates with IRF4 and cellular BATF (basic leucine zipper ATF-like TF) to drive a super-enhancer (SE)-mediated oncogenic transcriptional program in PEL. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) experiments demonstrated that IRF4, vIRF3, and BATF cooccupy the SEs of key survival genes, in a pattern that is distinct from those seen with other IRF4-driven malignancies. All three proteins cooperatively drive SE-mediated IRF4 overexpression. Inactivation of vIRF3 and, to a lesser extent, BATF phenocopies the gene expression changes and loss of cellular viability observed upon inactivation of IRF4. In sum, this work suggests that KSHV vIRF3 and cellular IRF4 and BATF cooperate as oncogenic transcription factors on SEs to promote cellular survival and proliferation in KSHV-associated lymphomas.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lymphoma (PEL). Here, we show that a viral transcription factor (vIRF3) cooperates with the cellular transcription factor IRF4 to control an oncogenic gene expression program in PEL cells. These proteins promote KSHV-mediated B cell transformation by activating the expression of prosurvival genes through super-enhancers. Our report thus demonstrates that this DNA tumor virus encodes a transcription factor that functions with cellular IRF4 to drive oncogenic transcriptional reprogramming.

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.

Genetic Analyses of Contributions of Viral Interleukin-6 Interactions and Signaling to Human Herpesvirus 8 Productive Replication.

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) is a cytokine that is poorly secreted and localized largely to the endoplasmic reticulum (ER). It has been implicated, along with other HHV-8 proinflammatory and/or angiogenic viral proteins, in HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), in addition to an MCD-related disorder involving systemic elevation of proinflammatory cytokines, including vIL-6 and human IL-6 (hIL-6). In these diseases, lytic (productive) replication, in addition to viral latency, is believed to play a critical role. Proreplication activity of vIL-6 has been identified experimentally in PEL and endothelial cells, but the relative contributions of different vIL-6 interactions have not been established. Productive interactions of vIL-6 with the IL-6 signal transducer, gp130, can occur within the ER, but vIL-6 also interacts in the ER with a nonsignaling receptor called vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2), calnexin, and VKORC1v2- and calnexin-associated proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GlucII). Here, we report the systematic characterization of interaction-altered vIL-6 variants and the lytic phenotypes of recombinant viruses expressing selected variants. Our data identify the critical importance of vIL-6 and its ER-localized activity via gp130 to productive replication in inducible SLK (epithelial) cells, absence of detectable involvement of vIL-6 interactions with VKORC1v2, GlucII, or UGGT1, and the insufficiency and lack of direct contributory effects of extracellular signaling by vIL-6 or hIL-6. These findings, obtained through genetics-based approaches, complement and extend previous analyses of vIL-6 activity.IMPORTANCE Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) was the first viral IL-6 homologue to be identified. Experimental and clinical evidence suggests that vIL-6 is important for the onset and/or progression of HHV-8-associated endothelial-cell and B-cell pathologies, including AIDS-associated Kaposi's sarcoma and multicentric Castleman's disease. The protein is unusual in its poor secretion from cells and its intracellular activity; it interacts, directly or indirectly, with a number of proteins beyond the IL-6 signal transducer, gp130, and can mediate activities through these interactions in the endoplasmic reticulum. Here, we report the characterization with respect to protein interactions and signal-transducing activity of a panel of vIL-6 variants and utilization of HHV-8 mutant viruses expressing selected variants in phenotypic analyses. Our findings establish the importance of vIL-6 in HHV-8 productive replication and the contributions of individual vIL-6-protein interactions to HHV-8 lytic biology. This work furthers understanding of the biological significance of vIL-6 and its unique intracellular interactions.

The RNA quality control pathway nonsense-mediated mRNA decay targets cellular and viral RNAs to restrict KSHV.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved RNA decay mechanism that has emerged as a potent cell-intrinsic restriction mechanism of retroviruses and positive-strand RNA viruses. However, whether NMD is capable of restricting DNA viruses is not known. The DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that NMD restricts KSHV lytic reactivation. Leveraging high-throughput transcriptomics we identify NMD targets transcriptome-wide in PEL cells and identify host and viral RNAs as substrates. Moreover, we identified an NMD-regulated link between activation of the unfolded protein response and transcriptional activation of the main KSHV transcription factor RTA, itself an NMD target. Collectively, our study describes an intricate relationship between cellular targets of an RNA quality control pathway and KSHV lytic gene expression, and demonstrates that NMD can function as a cell intrinsic restriction mechanism acting upon DNA viruses.