BCBL-1 Cells Research Articles

Delivery of miR-34a-5p by Folic Acid-Modified β-Cyclodextrin-Grafted Polyethylenimine Copolymer Nanocarriers to Resist KSHV

Kaposi’s sarcoma-associated herpesvirus (KSHV) infection can cause a range of malignancies. The use of microRNA (miRNA) to combat viruses and inhibit tumor development has received increasing attention. However, it is difficult for free miRNA to enter into cells directly and to degrade easily. Therefore, the miRNA therapy alone often fails to achieve the expected effect. The selection of an appropriate delivery system for effective miRNA delivery and anti-KSHV is rarely considered. In this study, a β-cyclodextrin-grafted polyethylenimine (β-CD-PEI) was prepared by the chemical synthesis method, and folic acid was further coupled to a nanocarrier (β-CD-PEI-FA). Gel electrophoresis retardation experiments showed that β-CD-PEI-FA could combine with miR-34a-5p to form a drug-carrying nanocomplex β-CD-PEI-FA/miR-34a-5p, which effectively protected miR-34a-5p from serum and nuclease degradation. The particle size and potential were 203.13 ± 0.41 nm and 27.02 ± 0.72 mV, respectively, which were suitable for endocytosis. The cytotoxicity of the vector to KSHV-positive cells BCBL-1 and SK-RG cells was detected by the CCK-8 assay, which proved that the vector had good biosafety in vitro. Further cell experiments showed that β-CD-PEI-FA/miR-34a-5p not only had the ability to transport miR-34a-5p but also accelerated the G2-phase process of BCBL-1 and SK-RG cells and inhibited the proliferation of KSHV-infected cells. Most importantly, it can significantly reduce the expression levels of KSHV genes ORF26, K8.1A, and LANA. Therefore, the nanocarriers may be a promising antiviral drug delivery system with potential applications in the anti-KSHV therapy.

DExD/H Box Helicases DDX24 and DDX49 Inhibit Reactivation of Kaposi's Sarcoma Associated Herpesvirus by Interacting with Viral mRNAs.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that is the causative agent of primary effusion lymphoma and Kaposi’s sarcoma. In healthy carriers, KSHV remains latent, but a compromised immune system can lead to lytic viral replication that increases the probability of tumorigenesis. RIG-I-like receptors (RLRs) are members of the DExD/H box helicase family of RNA binding proteins that recognize KSHV to stimulate the immune system and prevent reactivation from latency. To determine if other DExD/H box helicases can affect KSHV lytic reactivation, we performed a knock-down screen that revealed DHX29-dependent activities appear to support viral replication but, in contrast, DDX24 and DDX49 have antiviral activity. When DDX24 or DDX49 are overexpressed in BCBL-1 cells, transcription of all lytic viral genes and genome replication were significantly reduced. RNA immunoprecipitation of tagged DDX24 and DDX49 followed by next-generation sequencing revealed that the helicases bind to mostly immediate-early and early KSHV mRNAs. Transfection of expression plasmids of candidate KSHV transcripts, identified from RNA pull-down, demonstrated that KSHV mRNAs stimulate type I interferon (alpha/beta) production and affect the expression of multiple interferon-stimulated genes. Our findings reveal that host DExD/H box helicases DDX24 and DDX49 recognize gammaherpesvirus transcripts and convey an antiviral effect in the context of lytic reactivation.

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57.

RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. No circRNA was detectable from the PAN region. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with eight exons and seven introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF57 inhibits splicing of both K15 introns 1 and 2. ORF70/K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70/K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70/K3 locus, we further confirmed ORF57 regulation of ORF70/K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70/K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.

Kaposi's sarcoma-associated herpes virus-derived microRNA K12-1 over-activates the PI3K/Akt pathway to facilitate cancer progression in HIV-related gastrointestinal Kaposi's sarcoma.

Kaposi's sarcoma-associated herpes virus (KSHV) initiate and accelerate the development of Kaposi's sarcoma (KS), and KSHV possesses many cancer-associated genes, including KSHV-derived microRNA miR-K12-1, which has been identified to be closely associated with KS progression. However, the detailed mechanisms by which miR-K12-1 facilitates HIV-related gastrointestinal KS development are still not fully delineated. This study strived to evaluate the effect of miR-K12-1 on the progression of HIV-related gastrointestinal KS. The expression levels of miR-K12-1 in HIV-related gastrointestinal KS tissues were determined by RT-qPCR. Proliferation and apoptosis were assessed by colony formation, CCK-8 and flow cytometry, respectively. The expression of all proteins was detected by Western blot. The in vivo effect of miR-K12-1 on the formation of a tumor was explored by using the mouse xenograft model. In this study, we uncovered that KSHV-miR-K12-1 was upregulated in HIV-related gastrointestinal KS tissues and associated with poor outcome in HIV-related gastrointestinal KS patients. Compared with the control group, after miR-K12-1 inhibitor transfection, BCBL-1 cell viability was decreased, and the cell apoptosis was significantly increased, whereas transfection of miR-K12-1 mimics promoted cell proliferation and mitosis. In addition, our rescuing experiments verified that miR-K12-1 promoted cell proliferation via activating the PI3K/Akt pathway, and inhibition of the PI3K/Akt pathway by LY294002 abrogated the tumor-promoting effects of miR-K12-1 in HIV-related gastrointestinal KS. In summary, we concluded that KSHV-derived miR-K12-1 activate the PI3K/Akt pathway to initiate and accelerate the development of KS, which convinces us that miR-K12-1 can be used as potential biomarkers for KS diagnosis, treatment and prognosis.

Hesperetin inhibits KSHV reactivation and is reversed by HIF1α overexpression.

Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus, has two life cycle modes: the latent and lytic phases. KSHV lytic reactivation is important for both viral propagation and KSHV-induced tumorigenesis. The KSHV replication and transcription activator (RTA) protein is essential for lytic reactivation. Hesperetin, a citrus polyphenolic flavonoid, has antioxidant, anti-inflammatory, hypolipidemic, cardiovascular and anti-tumour effects. However, the effects of hesperetin on KSHV replication and KSHV-induced tumorigenesis have not yet been reported. Here, we report that hesperetin induces apoptotic cell death in BCBL-1 cells in a dose-dependent manner. Hesperetin inhibits KSHV reactivation and reduces the production of progeny virus from KSHV-harbouring cells. We also confirmed that HIF1α promotes the RTA transcriptional activities and lytic cycle-refractory state of KSHV-infected cells. Hesperetin suppresses HIF1α expression to inhibit KSHV lytic reactivation. These results suggest that hesperetin may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.

Arsenic Trioxide and Lenalidomide: A Promising Combination Therapy for Primary Effusion Lymphoma

Primary Effusion Lymphoma (PEL) is an aggressive malignancy caused by the Kaposi sarcoma associated herpesvirus (KSHV). PEL patients rarely respond to conventional chemotherapy and their prognosis is poor, with a median survival of less than six months. The most promising treatment strategies in these patients combine anti-viral treatments with new agents including chemicals and cytokines. Here, we investigated the efficacy of lenalidomide (Lena), an immune modulator with significant clinical activity in a large number of hematological malignancies, and arsenic trioxide (As), a potent agent in acute promyelocytic leukemia and HTLV-I associated adult T cell leukemia.We used three PEL cell lines (BC-1, BC-3 and BCBL-1 which are KSHV+/EBV- malignant B cells derived from PEL patients) and one PEL-negative control cell line (RAJI as a KSHV- malignant B cell line). We demonstrate that the combination of As and Lena resulted in growth inhibition in BC-1 and BC-3 cells and to a lesser degree in BCBL-1 cells while no effect was observed in RAJI cells. This growth arrest was associated with induction of massive apoptosis and a substantial upregulation of phosphorylated and total p53 protein levels. Furthermore, As/Lena inhibited the expression of latent viral transcripts (LANA-1, LANA-2, v-FLIP and v-Cyclin) and proteins (LANA-1/LANA-2) in all tested PEL cells.Finally, and while Lena alone did not have any inhibitory effect on in vitro treated PEL cells, single agent Lena treatment doubled survival of BC-3 PEL mice and significantly prolonged survival in 75% of BCBL-1 PEL mice and led to cure of the remaining 25%. More importantly, combination of As and Lena was highly synergistic and cured 25% and 75% of BC-3 and BCBL-1 PEL mice respectively. These results provide a rational for the therapeutic use of the combination As/Lena in PEL patients. DisclosuresNo relevant conflicts of interest to declare.

Establishment of Nude Mice Lacking NK Cells and Their Application for Human Tumor Xenografts

Objective:Nude mice are used as a recipient for human tumor cell xenografts. However, the success rate of xenotransplantation is unsatisfactory due to high natural killer (NK) activity. To overcome this limitation, we established nude mice with no NK cells, and compared the transplantation efficiency with that in nude mice.Methods: BALB/c Nude Jak3-/- (Nude-J) mice were established by crossing BALB/c Nude mice and BALB/c Jak-3-/- mice. Hematopoietic malignant cell lines (BCBL-1 and Z138) were implanted subcutaneously to compare the tumorigenicity in Nude-J mice with Nude Rag-2/Jak3 double deficient (Nude RJ) mice and nude mice. Results:Nude-J mice showed complete loss of NK and T lymphocytes, whereas B lymphocytes remained. Both BCBL-1 and Z138 human lymphoid malignant cell lines formed almost the same sizes of subcutaneous tumors in Nude-J and Nude RJ mice, whereas they formed no or only small tumors in nude mice. Splenocytes from Nude-J mice showed no cytotoxic activity in vitro. Conclusion:Nude-J mice can be a valuable tool for human tumor cell transplantation studies.

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

BackgroundPrimary 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. PurposeTo 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 designWe evaluated the anti-cancer activities of CuB in vitro and in vivo. MethodsCell 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. ResultsCuB 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. ConclusionThis study suggests that CuB is a promising agent for PEL treatment.

Gallic acid inhibits Kaposi's Sarcoma-associated herpesvirus lytic reactivation by suppressing RTA transcriptional activities.

Kaposi's sarcoma‐associated herpesvirus (KSHV), an oncogenic virus, has two life cycle modes: the latent and lytic phases. KSHV lytic reactivation is known to be important both for viral propagation and for KSHV‐induced tumorigenesis. The KSHV replication and transcription activator (RTA) protein is essential for lytic reactivation. Gallic acid (GA), one of the most abundant phenolic acids in the plant kingdom, has been shown potential chemotherapeutic efficacy against microbial and cancer. However, the effects of GA on KSHV replication and KSHV‐induced tumorigenesis have not yet been reported. Here, we report that GA induces apoptotic cell death in BCBL‐1 cells in a dose‐dependent manner. GA inhibits KSHV reactivation and reduces the production of progeny virus from KSHV‐harboring cells. GA inhibits RTA transcriptional activities by suppressing its binding to target gene promoters. These results suggest that GA may represent a novel strategy for the treatment of KSHV infection and KSHV‐associated lymphomas.

Abstract 1394: Gene expression profile in BCBL-1 cell line following activation of KSHV

Abstract Patients with HIV/AIDS are at a 3,400 times higher risk of developing Kaposi's sarcoma when compared to healthy populations. Our hypothesis is that the upregulation of viral associated proteins is causing an increase in DNA damage, allowing for the higher incidence rate of Kaposi's sarcoma in infected populations. The direct mechanisms leading to this increased risk is unknown, but the viral infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is an apparent cause of this risk. The cell is under attack of endogenous threats including oxidative stress, causing DNA damage and leading to mutations, but the viral proteins may interfere with the repair mechanisms. In previous experiments we looked at DNA repair dynamics following X-ray exposure in the two different stages and determined there is an inefficiency in the DNA repair process in the cells during active production of viral particles. This project aimed to compare the transcriptomes of the BCBL-1 cell line during latent and acute phases of KSHV production. Our results are showing that genes involved in genome stability are differently expressed during the lytic stage. Comparison of these two transcriptomes allows for identification of which proteins are being upregulated during active viral production. This may be of importance for the HIV/AIDS cancer patients because it shows the mechanisms behind genome stability in KSHV-infected cells. 1) Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 2) NASA EPSCoR, NNH15ZHA003C Citation Format: Samantha L. Haines, Sabrina R. Bishop, Viktorija Podlutskaya, Andrej Podlutsky. Gene expression profile in BCBL-1 cell line following activation of KSHV [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1394.

P300 promotes cell proliferation through suppressing Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in the infected B-lymphoma cells

Primary Effusion Lymphoma (PEL) is a B-cell lymphoma associated with Kaposi's sarcoma herpesvirus (KSHV) infection. However, the mechanism of oncogenesis of PEL is still unclear. Studies have shown that the cellular transcriptional coactivator p300 regulates the interaction between host and virus, which plays a vital role in viral replication. In this study, we investigated the role of p300 in BCBL1 cells during the KSHV life cycle. We found that p300 knockout resulted in an overall increase for the early lytic genes and changed the expression of genes associated with tumor development, proliferation, and the immune response in the KSHV infected B cells. However, knockout of p300 significantly inhibited the expression of the immediate-early gene RTA and the late lytic gene K8 after KSHV lytic activation. Additionally, the intracellular KSHV genome copy number and the virion production were reduced. These results demonstrated that p300 plays a crucial role in suppressing KSHV viral replication in BCBL1. Furthermore, we observed that the growth of BCBL1 was inhibited by knockout of p300, which confirmed our findings that p300 promotes cell proliferation. This study further provided evidence that p300 plays an important role in the pathogenesis of BCBL1, which might lead to the oncogenesis of PEL caused by KSHV infection.

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.

Identification of new antiviral agents against Kaposi's sarcoma-associated herpesvirus (KSHV) by high-throughput drug screening reveals the role of histamine-related signaling in promoting viral lytic reactivation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes several human cancers, such as Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). Current treatment options for KSHV infection and virus associated diseases are sometimes ineffective, therefore, more effectively antiviral agents are urgently needed. As a herpesvirus, lytic replication is critical for KSHV pathogenesis and oncogenesis. In this study, we have established a high-throughput screening assay by using an inducible KSHV+ cell-line, iSLK.219. After screening a compound library that consisted of 1280 Food and Drug Administration (FDA)-approved drugs, 15 hit compounds that effectively inhibited KSHV virion production were identified, most of which have never been reported with anti-KSHV activities. Interestingly, 3 of these drugs target histamine receptors or signaling. Our data further confirmed that antagonists targeting different histamine receptors (HxRs) displayed excellent inhibitory effects on KSHV lytic replication from induced iSLK.219 or BCBL-1 cells. In contrast, histamine and specific agonists of HxRs promoted viral lytic replication from induced iSLK.219 or KSHV-infected primary cells. Mechanistic studies indicated that downstream MAPK and PI3K/Akt signaling pathways were required for histamine/receptors mediated promotion of KSHV lytic replication. Direct knockdown of HxRs in iSLK.219 cells effectively blocked viral lytic gene expression during induction. Using samples from a cohort of HIV+ patients, we found that the KSHV+ group has much higher levels of histamine in their plasma and saliva than the KSHV- group. Taken together, our data have identified new anti-KSHV agents and provided novel insights into the molecular bases of host factors that contribute to lytic replication and reactivation of this oncogenic herpesvirus.

KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication.

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.

DNA Repair Process in Virally Infected Cells

Humans come into regular contact with a spectrum of infectious agents, such as viruses. According to the CDC, there are over 200 viruses that contribute to just the common cold. Viruses will infect host cell DNA and begin to produce new viruses. In addition to viral challenges, cells must defend against endogenous threats such as oxidative stress, which cause a variety of DNA damage and could lead to mutations. These and other stimuli result in cellular efforts to repair DNA, but sometimes the damage is not repaired properly. This may occur because of viruses hijacking the DNA repair machinery while cells attempt to repair their DNA. Our hypothesis is that the virus‐infected cells could not repair their damaged DNA properly and as a result, damage is passed onto subsequent generations in the form of mutations. Accumulation of damaged and mutated DNA results in a predisposition to cancer and subsequent progression to tumor formation. For example, individuals infected with HIV/AIDS, caused by a virus, show a greater increase in certain types of cancer. Using Human Herpes Virus 8, the show the link between virus infections and cellular DNA repair function. Through measuring DNA repair with the Comet Assay, and by analyzing the level of apoptosis, we conclude that active production HHV8 in BCBL1 cells interfere with the DNA repair of X‐ray and UV‐C induced DNA damage.Support or Funding Information1) Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual: www.alaska.edu/titleIXcompliance/nondiscrimination.2) NASA EPSCoR, NNH15ZHA003CThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Tip60 Reduces Lytic and Latent Gene Expression of Kaposi's Sarcoma-Associated Herpes Virus (KSHV) and Proliferation of KSHV-Infected Tumor Cells.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi’s sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman’s disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60’s role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV+/EBV- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics.

Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation and de novo infection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UV cross-linking and immunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication during de novo infection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during de novo infection.IMPORTANCE Genomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.

Effects of cyclin-dependent kinase 9 (CDK9) on lytic replication of Kaposi′s sarcoma-associated herpesvirus (KSHV)

Objective To investigate the role of cyclin-dependent kinase 9 (CDK9) in regulating lytic replication of Kaposi′s sarcoma-associated herpesvirus (KSHV). Methods Percentages of red fluorescent protein (RFP) positive cells were counted after treating and stimulating iSLK.219 cells with FIT-039, a CDK9 specific inhibitor, and doxycycline (Dox), respectively, or transfecting and stimulating them with CDK9 siRNA (si-CDK9) and Dox, respectively. Quantitative real-time PCR was used to detect the expression of KSHV genes at mRNA level in TREx-K-Rta BCBL-1 cells treated with FIT-039 or transfected with si-CKD9 in the presence of Dox. Chromatin immunoprecipitation (ChIP) assay was used to examine the enrichment of RNA polymerase Ⅱ (RNA Pol Ⅱ) and phosphorylated CTD-S2 of RNA Pol Ⅱ on the PAN promoter in TREx-K-Rta BCBL-1 cells treated with FIT-039. Results Both FIT-039 intervention and CDK9 knockdown dramatically deceased the percentage of RFP-positive iSLK.219 cells and suppressed the expression of ORF50, PAN and K2 in TREx-K-Rta BCBL-1 cells at mRNA level. Furthermore, FIT-039 significantly inhibited the enrichment of RNA Pol Ⅱ and phosphorylated CTD-S2 of RNA Pol Ⅱ on the PAN promoter in TREx-K-Rta BCBL-1 cells. Conclusion CDK9 is involved in regulating the lytic replication of KSHV through promoting the phosphorylation of CTD-S2 of RNA Pol Ⅱ. Key words: Cyclin-dependent kinase 9; Kaposi′s sarcoma-associated herpesvirus; Lytic replication; Gene transcriptional elongation

Restoring PU.1 induces apoptosis and modulates viral transactivation via interferon-stimulated genes in primary effusion lymphoma.

Primary effusion lymphoma (PEL), which is an aggressive subgroup of B-cell lymphoma associated with Kaposi sarcoma-associated herpes virus/human herpes virus-8, is refractory to the standard treatment, and exhibits a poor survival. Although PU.1 is downregulated in PEL, the potential role of its reduction remains to be elucidated. In this investigation, we analyzed the DNA methylation of PU.1 cis-regulatory elements in PEL and the effect of restoring PU.1 on PEL cells. The mRNA level of PU.1 was downregulated in PEL cells. The methylated promoter and enhancer regions of the PU.1 gene were detected in PEL cells. Suppression of cell growth and apoptosis were caused by the restoration of PU.1 in PEL cells. A microarray analysis revealed that interferon-stimulated genes (ISGs) including pro-apoptotic ISGs were strongly increased in BCBL-1 cells after the induction of PU.1. Reporter assays showed that PU.1 transactivated pro-apoptotic ISG promoters, such as the XAF1, OAS1 and TRAIL promoters. Mutations at the PU.1 binding sequences suppressed its transactivation. We confirmed the binding of PU.1 to the XAF1, OAS1 and TRAIL promoters in a chromatin immunoprecipitation assay. PU.1 suppressed ORF57 activation by inducing IRF7. The reinduction of PU.1 reduced formation of ascites and lymphoma cell infiltration of distant organs in PEL xenograft model mice. Collectively, PU.1 has a role in tumor suppression in PEL and its down-regulation is associated with PEL development. Restoring PU.1 with demethylation agents may be a novel therapeutic approach for PEL.

NLRX1 negatively modulates type I IFN to facilitate KSHV reactivation from latency.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a herpesvirus that is linked to Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV establishes persistent latent infection in the human host. KSHV undergoes periods of spontaneous reactivation where it can enter the lytic replication phase of its lifecycle. During KSHV reactivation, host innate immune responses are activated to restrict viral replication. Here, we report that NLRX1, a negative regulator of the type I interferon response, is important for optimal KSHV reactivation from latency. Depletion of NLRX1 in either iSLK.219 or BCBL-1 cells significantly suppressed global viral transcription levels compared to the control group. Concomitantly, fewer viral particles were present in either cells or supernatant from NLRX1 depleted cells. Further analysis revealed that upon NLRX1 depletion, higher IFNβ transcription levels were observed, which was also associated with a transcriptional upregulation of JAK/STAT pathway related genes in both cell lines. To investigate whether IFNβ contributes to NLRX1’s role in KSHV reactivation, we treated control and NLRX1 depleted cells with a TBK1 inhibitor (BX795) or TBK1 siRNA to block IFNβ production. Upon BX795 or TBK1 siRNA treatment, NLRX1 depletion exhibited less inhibitory effects on reactivation and infectious virion production, suggesting that NLRX1 facilitates KSHV lytic replication by negatively regulating IFNβ responses. Our data suggests that NLRX1 plays a positive role in KSHV lytic replication by suppressing the IFNβ response during the process of KSHV reactivation, which might serve as a potential target for restricting KSHV replication and transmission.