Kaposi's Sarcoma-associated Herpesvirus Replication Research Articles

Expression and Function of Kaposi’s Sarcoma-Associated Herpesvirus Non-coding RNAs

Herpesviruses are large DNA viruses whose genomes contain a number of genes producing non-coding RNAs, such as small RNA and long non-coding RNA. In this review, the expression and function of Kaposi’s sarcoma-associated herpesvirus (KSHV) non-coding RNAs are discussed. The KSHV genome contains at least 12 genes from which pre-microRNAs (miRNAs) are transcribed, and several genes producing long non-coding RNAs (lncRNAs). Polyadenylated nuclear RNA is a highly expressed viral lncRNA molecule in KSHV-infected cells and is necessary for KSHV replication. KSHV miRNAs are expressed predominantly during the latent phase of KSHV infection not only in cells but also in extracellular vesicles such as exosomes and virions. Identification of viral miRNA targets has revealed that KSHV miRNAs have various functions relating to oncogenesis, anti-apoptosis, angiogenesis, and migration of KSHV-infected cells. Some KSHV miRNAs regulate KSHV replication to maintain viral latency. KSHV non-coding RNAs play significant roles in the pathogenesis of KSHV-associated diseases.

Repurposing Cytarabine for Treating Primary Effusion Lymphoma by Targeting Kaposi's Sarcoma-Associated Herpesvirus Latent and Lytic Replications.

ABSTRACTOncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) is etiologically linked to primary effusion lymphoma (PEL), an aggressive and nontreatable malignancy commonly found in AIDS patients. In this study, we performed a high-throughput screening of 3,731 characterized compounds and identified cytarabine, approved by the FDA for treating numerous types of cancer, as a potent inhibitor of KSHV-induced PEL. We showed the high efficacy of cytarabine in the growth inhibition of various PEL cells by inducing cell cycle arrest and apoptosis. Cytarabine inhibited host DNA and RNA syntheses and therefore induced cellular cytotoxicity. Furthermore, cytarabine inhibited viral DNA and RNA syntheses and induced the rapid degradation of KSHV major latent protein LANA (latency-associated nuclear antigen), leading to the suppression of KSHV latent replication. Importantly, cytarabine effectively inhibited active KSHV replication and virion production in PEL cells. Finally, cytarabine treatments not only effectively inhibited the initiation and progression of PEL tumors but also induced regression of grown PEL tumors in a xenograft mouse model. Altogether, our study has identified cytarabine as a novel therapeutic agent for treating PEL as well as eliminating KSHV persistent infection.

Caspase-Dependent Suppression of Type I Interferon Signaling Promotes Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication

An important component of lytic infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is the ability of the virus to evade the innate immune response, specifically type I interferon (IFN) responses that are triggered by recognition of viral nucleic acids. Inhibition of type I IFN responses by the virus promotes viral replication. Here, we report that KSHV uses a caspase-dependent mechanism to block type I IFN, in particular IFN-β, responses during lytic infection. Inhibition of caspases during KSHV reactivation resulted in increased TBK1/IKKε-dependent phosphorylation of IRF3 as well as elevated levels of IFN-β transcription and secretion. The increased secretion of IFN-β upon caspase inhibition reduced viral gene expression, viral DNA replication, and virus production. Blocking IFN-β production or signaling restored viral replication. Overall, our results show that caspase-mediated regulation of pathogen sensing machinery is an important mechanism exploited by KSHV to evade innate immune responses.IMPORTANCE KSHV is the causative agent of Kaposi's sarcoma (KS), an AIDS-defining tumor that is one of the most common causes of cancer death in sub-Saharan Africa. In this study, we examined the role of a set of cellular proteases, called caspases, in the regulation of immune responses during KSHV infection. We demonstrate that caspases prevent the induction and secretion of the antiviral factor IFN-β during replicative KSHV infection. The reduced IFN-β production allows for high viral gene expression and viral replication. Therefore, caspases are important for maintaining KSHV replication. Overall, our results suggest that KSHV utilizes caspases to evade innate immune responses, and that inhibiting caspases could boost the innate immune response to this pathogen and potentially be a new antiviral strategy.

KSHV episomes reveal dynamic chromatin loop formation with domain-specific gene regulation

The three-dimensional structure of chromatin organized by genomic loops facilitates RNA polymerase II access to distal promoters. The Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic transcriptional program is initiated by a single viral transactivator, K-Rta. Here we report the KSHV genomic structure and its relationship with K-Rta recruitment sites using Capture Hi–C analyses. High-resolution 3D viral genomic maps identify a number of direct physical, long-range, and dynamic genomic interactions. Mutant KSHV chromosomes harboring point mutations in the K-Rta responsive elements (RE) significantly attenuate not only the directly proximate downstream gene, but also distal gene expression in a domain-specific manner. Genomic loops increase in the presence of K-Rta, while abrogation of K-Rta binding impairs the formation of inducible genomic loops, decreases the expression of genes networked through the looping, and diminishes KSHV replication. Our study demonstrates that genomic architectural dynamics plays an essential role in herpesvirus gene expression.

IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) causes a persistent infection, presenting latent and lytic replication phases during its life cycle. KSHV-related diseases are associated with deregulated expression of inflammatory cytokines, including IL-6 and IL-10, but the mechanisms underlying this dysregulation are unclear. Herein, we report a molecular mechanism for KSHV-induced IL-10 gene expression. KSHV replication and transcription activator (K-RTA) is a molecular switch for the initiation of expression of viral lytic genes, and we describe, for the first time, that K-RTA significantly activates the promoter of the human IL-10 gene. Of note, mutations involving a basic region of K-RTA reduced the association of K-RTA with the IL-10 promoter. Moreover, the host-cell transcription factors, specificity proteins (SP) 1 and 3, play a pivotal cooperative role in K-RTA-mediated transactivation of the IL-10 promoter. K-RTA can interact with SP1 and SP3 directly in vitro, and electrophoresis mobility shift assays (EMSAs) revealed co-operative interaction involving K-RTA, SP1, and SP3 in binding to the IL-10 promoter. As DNase I footprinting assays indicated that K-RTA did not affect SP3 binding to the IL-10 promoter, SP3 can function to recruit K-RTA to the IL-10 promoter. These findings indicate that K-RTA can directly contribute to IL-10 up-regulation via a functional interplay with the cellular transcription factors SP1 and SP3.

KSHV Genome Replication and Maintenance in Latency.

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), is the eighth human herpesvirus found by Yuan Chang and Patrick Moore, 1992. It is a Rhadinovirus belonging to the gamma herpesvirus subfamily. As known for many gamma herpesviruses, KSHV is also well-correlated to several cancer formations such as Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Different from the other herpesvirus subfamily, gamma herpesviruses establish latency as a default infection strategy when they infect to the target cells, as KSHV is present as the latent form in the related cancers. In the latency, the virus expresses a limited number of the genes such as latency-associated nuclear antigen (LANA), v-cyclin (v-CYC, ORF72), v-FLIP (K13), kaposin (K12), and 25 microRNAs (K-miRNAs). The virus replicates according to cellular replication machinery with a viral replication origin (ori-P) and LANA. Then, the replicated genome is segregated equally to daughter cells by appearance to maintain the virus genome copy number per cell. The virus makes the most use of cellular machinery to achieve this end. In this chapter, I would like to review KSHV replication and gene expression in the latency and discuss.

Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer that commonly affects patients with AIDS and which is endemic in sub-Saharan Africa. The KSHV capsid is highly pressurized by its double-stranded DNA genome, as are the capsids of the eight other human herpesviruses. Capsid assembly and genome packaging of herpesviruses are prone to interruption and can therefore be targeted for the structure-guided development of antiviral agents. However, herpesvirus capsids-comprising nearly 3,000 proteins and over 1,300 Å in diameter-present a formidable challenge to atomic structure determination and functional mapping of molecular interactions. Here we report a 4.2 Å resolution structure of the KSHV capsid, determined by electron-counting cryo-electron microscopy, and its atomic model, which contains 46 unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP) and the triplex proteins Tri1 and Tri2. Our structure and mutagenesis results reveal a groove in the upper domain of the MCP that contains hydrophobic residues that interact with the SCP, which in turn crosslinks with neighbouring MCPs in the same hexon to stabilize the capsid. Multiple levels of MCP-MCP interaction-including six sets of stacked hairpins lining the hexon channel, disulfide bonds across channel and buttress domains in neighbouring MCPs, and an interaction network forged by the N-lasso domain and secured by the dimerization domain-define a robust capsid that is resistant to the pressure exerted by the enclosed genome. The triplexes, each composed of two Tri2 molecules and a Tri1 molecule, anchor to the capsid floor via a Tri1 N-anchor to plug holes in the MCP network and rivet the capsid floor. These essential roles of the MCP N-lasso and Tri1 N-anchor are verified by serial-truncation mutageneses. Our proof-of-concept demonstration of the use of polypeptides that mimic the smallest capsid protein to inhibit KSHV lytic replication highlights the potential for exploiting the interaction hotspots revealed in our atomic structure to develop antiviral agents.

Arctigenin induces the apoptosis of primary effusion lymphoma cells under conditions of glucose deprivation.

Kaposi's sarcoma-associated herpesvirus(KSHV) is the causative agent of primary effusion lymphoma(PEL) and Kaposi's sarcoma. PEL is a type of non-Hodgkin's B-cell lymphoma, affecting immunosuppressed individuals, such as post-transplant or AIDS patients. However, since PEL is resistant to chemotherapeutic regimens, new effective treatment strategies are required. Arctigenin, a natural lignan compound found in the plant Arctium lappa, has been widely investigated as a potential anticancer agent in the clinical setting. In the present study, we examined the cytotoxic effects of arctigenin by cell viability assay and found that arctigenin markedly inhibited the proliferation of PEL cells compared with KSHV-uninfected B-lymphoma cells under conditions of glucose deprivation. Arctigenin decreased cellular ATP levels, disrupted mitochondrial membrane potential and triggered caspase-9-mediated apoptosis in the glucose-deprived PEL cells. In addition, western blot analysis using phospho-specific antibodies were used to evaluate activity changes in the signaling pathways of interest. As a result, arctigenin suppressed the activation of the extracellular signal-regulated kinase(ERK) and p38mitogen-activated protein kinase (p38MAPK) signaling pathways by inhibiting ERK and p38MAPK phosphorylation in the glucose-deprived PEL cells. We confirmed that an inhibitor of ERK (U0126) or p38MAPK (SB202190 and SB203580) suppressed the proliferation of the BC3 PEL cells compared with the KSHV-negative DG75 cells. Moreover, RT-PCR and luciferase reporter assay revealed that arctigenin and p38MAPK inhibition by SB202190 or SB203580 downregulated the transcriptional expression of unfolded protein response(UPR)‑related molecules, including GRP78 and ATF6α under conditions of glucose deprivation. Finally, we confirmed that arctigenin did not affect KSHV replication in PEL cells, suggesting that arctigenin treatment for PEL does not contribute to the risk of denovo KSHV production. These data thus indicate that arctigenin may serve as a lead compound for the development of novel and effective drugs for the treatment of PEL.

Localization of Double-Strand Break Repair Proteins to Viral Replication Compartments following Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

ABSTRACTDouble-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance amplification of viral DNA. Although the recruitment of DSB-sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event, indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signaling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA-packaging proteins provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication.IMPORTANCE Tumor viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localization of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB-sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection.

ZIC2 Is Essential for Maintenance of Latency and Is a Target of an Immediate Early Protein during Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation.

ABSTRACTBivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression, while the H3K4me3 mark prevents the region from becoming permanently silenced and prepares the domain for activation when needed. Specific regions of Kaposi's sarcoma-associated herpesvirus (KSHV) latent episomes are poised to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function; thus, we hypothesized that identification of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication. Here we show that a zinc finger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquitinate ZIC2. ZIC2 localized at immediate early and early gene cluster regions of the KSHV genome and contributed to tethering of polycomb repressive complex 2 through physical interaction, thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted the balance of bivalent histone modifications toward more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication.IMPORTANCE Posttranslational histone modifications regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reactivatable by K-Rta remains unclear. We found that ZIC2, a transcriptional factor essential for stem cell pluripotency, plays a role in maintaining KSHV latent infection in naturally infected cells. We found that ZIC2 degradation by K-Rta shifts bivalent histone marks to a more active configuration, leading to KSHV reactivation. ZIC2 interacts with and maintains polycomb repressor complex 2 at the K-Rta promoter. Our findings uncover (i) a mechanism utilized by KSHV to maintain latent infection, (ii) a latency-lytic cycle switch operated by K-Rta, and (iii) a molecular mechanism of ZIC2-mediated local histone modification.

1, 25(OH)2 D3 Induces Reactivation and Death of Kaposi\u2019s Sarcoma-Associated Herpesvirus of Primary Effusion Lymphoma cells

Kaposi’s sarcoma associated herpesvirus (KSHV) a gammaherpesvirus establishes perennial latency in the host with periodic reactivation. Occasionally change in the physiological condition like hypoxia, host cell differentiation can trigger the lytic switch and reactivation of the virus. The biologically active form of 1, 25(OH)2 D3 plays a critical role in the regulation of various physiological processes (e.g. regulation of mineral homeostasis and control of bone metabolism). Apart from its role in host physiology, 1, 25(OH)2 D3 has been implicated as a potential agent for the prevention and/or treatment of many a tumors. Here we show that 1, 25(OH)2 D3 induces both death of Kaposi sarcoma associated herpesvirus infected PEL cells and KSHV replication. 1, 25(OH)2 D3 mediated inhibition of proliferation was associated with apoptosis of the PEL cells, and virus reactivation. In addition, p38 signalling is required for KSHV reactivation. Furthermore, treatment of PEL cells with p38 inhibitor abrogated the expression of ORF57, thus blocking lytic switch. Furthermore, silencing of VDR resulted in reduced ORF57 expression compared to the control cells, signifying the potential role of 1, 25(OH)2 D3 in KSHV reactivation. Thus, our studies have revealed a novel role of 1, 25(OH)2 D3 in the regulation of KSHV reactivation and PEL cell death.

Virus-Like Vesicles of Kaposi's Sarcoma-Associated Herpesvirus Activate Lytic Replication by Triggering Differentiation Signaling.

Virus-like vesicles (VLVs) are membrane-enclosed vesicles that resemble native enveloped viruses in organization but lack the viral capsid and genome. During the productive infection of tumor-associated gammaherpesviruses, both virions and VLVs are produced and are released into the extracellular space. However, studies of gammaherpesvirus-associated VLVs have been largely restricted by the technical difficulty of separating VLVs from mature virions. Here we report a strategy of selectively isolating VLVs by using a Kaposi's sarcoma-associated herpesvirus (KSHV) mutant that is defective in small capsid protein and is unable to produce mature virions. Using mass spectrometry analysis, we found that VLVs contained viral glycoproteins required for cellular entry, as well as tegument proteins involved in regulating lytic replication, but lacked capsid proteins. Functional analysis showed that VLVs induced the expression of the viral lytic activator RTA, initiating KSHV lytic gene expression. Furthermore, employing RNA sequencing, we performed a genomewide analysis of cellular responses triggered by VLVs and found that PRDM1, a master regulator in cell differentiation, was significantly upregulated. In the context of KSHV replication, we demonstrated that VLV-induced upregulation of PRDM1 was necessary and sufficient to reactivate KSHV by activating its RTA promoter. In sum, our study systematically examined the composition of VLVs and demonstrated their biological roles in manipulating host cell responses and facilitating KSHV lytic replication.IMPORTANCE Cells lytically infected with tumor-associated herpesviruses produce a high proportion of virus-like vesicles (VLVs). The composition and function of VLVs have not been well defined, largely due to the inability to efficiently isolate VLVs that are free of virions. Using a cell system capable of establishing latent KSHV infection and robust reactivation, we successfully isolated VLVs from a KSHV mutant defective in the small capsid protein. We quantitatively analyzed proteins and microRNAs in VLVs and characterized the roles of VLVs in manipulating host cells and facilitating viral infection. More importantly, we demonstrated that by upregulating PRDM1 expression, VLVs triggered differentiation signaling in targeted cells and facilitated viral lytic infection via activation of the RTA promoter. Our study not only demonstrates a new strategy for isolating VLVs but also shows the important roles of KSHV-associated VLVs in intercellular communication and the viral life cycle.

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.

KSHV co-infection down-regulates HPV16 E6 and E7 from cervical cancer cells.

High-risk human papillomavirus (HPV) infection is the etiological agent of some malignancies such as cervical, oral and oropharyngeal cancers. Kaposi sarcoma-associated herpesvirus (KSHV) represents a principal causative agent of several human cancers arising in those immunocompromised patients. Interestingly, KSHV DNA has been detected in the oral cavity and the female genital tract, although its detection rate in cervical samples is very low and few reports are about KSHV/HPV co-infection. Therefore, it remains unclear about the role of KSHV co-infection in the development of HPV-related neoplasias. In the current study, we report that HPV16-integrated cervical cancer cell-line SiHa is susceptible to KSHV latent infection and replication. We also have found that KSHV infection or viral latent proteins are capable of reducing HPV16 E6/E7 expression through the manipulation of cellular microRNA function. Array analysis indicates that KSHV infection induces some inflammatory cytokines/chemokines production as well as up-regulates a series of interferon-induced genes expression, which may facilitate host immune defense system attacking these co-infected cells and clearance of viruses. Together, our data have provided possible explanations for very low detection rate of KSHV shedding as well as of KSHV/HPV co-infection in cervical samples and/or cervical cancer cells.

The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication.

KS-Bcl-2 is a Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Bcl-2 (vBcl-2) homolog which has apoptosis- and autophagy-inhibiting activity when expressed in transfected cells. However, little is known about its function during viral infection. As KS-Bcl-2 is expressed during the lytic replication cycle, we used constitutively lytic and inducibly lytic KSHV mutants to investigate the role of KS-Bcl-2 during the lytic cycle. We show that KSHV cannot complete the lytic replication cycle and produce infectious progeny in the absence of KS-Bcl-2, indicating that the protein is essential for KSHV replication. Replacement of the KS-Bcl-2 coding sequence, ORF16, by sequences encoding a potent cellular apoptosis and autophagy inhibitor, Bcl-XL, or the cytomegalovirus mitochondrial inhibitor of apoptosis, vMIA, did not rescue KSHV replication, suggesting that KS-Bcl-2 has a function that goes beyond apoptosis and autophagy inhibition. Strikingly, the vBcl-2 proteins of the related γ2-herpesviruses murine herpesvirus 68 and herpesvirus saimiri did not rescue the replication of a KS-Bcl-2 deletion mutant, but rhesus rhadinovirus (RRV) vBcl-2 did. Deletion of ORF16 from the RRV genome abrogated viral replication, but its replacement by KSHV ORF16 rescued RRV replication, indicating that the essential vBcl-2 function is conserved between these two primate rhadinoviruses. We further show that the KSHV and RRV Bcl-2 homologs localize to the mitochondria and nuclei of infected cells. Deletion of 17 amino acids from the N terminus of KS-Bcl-2 abrogates nuclear localization and KSHV replication, suggesting that KS-Bcl-2 might execute its essential function in the nuclei of infected cells.IMPORTANCE Several viruses express proteins homologous to cellular Bcl-2. Viral Bcl-2 proteins have functions similar to those of cellular Bcl-2: they can inhibit apoptosis, a form of programmed cell death, and autophagy, a self-degradative process for the disposal of dysfunctional or unwanted components. This study shows that the vBcl-2 proteins of KSHV and RRV differ from other vBcl-2 proteins in that they are essential for viral replication. The essential function is separate from the apoptosis- and autophagy-inhibiting activity but correlates with an unusual localization within the cell nucleus, suggesting that these proteins exert a novel function in the nucleus.

Niclosamide inhibits lytic replication of Epstein-Barr virus by disrupting mTOR activation

Infection with the oncogenic γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause several severe malignancies in humans. Inhibition of the lytic replication of EBV and KSHV eliminates the reservoir of persistent infection and transmission, consequently preventing the occurrence of diseases from the sources of infection. Antiviral drugs are limited in controlling these viral infectious diseases. Here, we demonstrate that niclosamide, an old anthelmintic drug, inhibits mTOR activation during EBV lytic replication. Consequently, niclosamide effectively suppresses EBV lytic gene expression, viral DNA lytic replication and virion production in EBV-infected lymphoma cells and epithelial cells. Niclosamide exhibits cytotoxicity toward lymphoma cells and induces irreversible cell cycle arrest in lytically EBV-infected cells. The ectopic overexpression of mTOR reverses the inhibition of niclosamide in EBV lytic replication. Similarly, niclosamide inhibits KSHV lytic replication. Thus, we conclude that niclosamide is a promising candidate for chemotherapy against the acute occurrence and transmission of infectious diseases of oncogenic γ-herpesviruses.

High Glucose Induces Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus

A high prevalence of Kaposi's sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological conditions of diabetes contribute to KS development. We found elevated levels of viral lytic gene expression when Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells were cultured in high-glucose medium. To demonstrate the association between high glucose levels and KSHV replication, we xenografted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV cells) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high glucose levels induced the production of hydrogen peroxide (H2O2), which downregulated silent information regulator 1 (SIRT1), a class III histone deacetylase (HDAC), resulting in the epigenetic transactivation of KSHV lytic genes. These results suggest that high blood glucose levels in diabetic patients contribute to the development of KS by promoting KSHV lytic replication and infection. IMPORTANCE Multiple epidemiological studies have reported a higher prevalence of classic KS in diabetic patients. By using both in vitro and in vivo models, we demonstrated an association between high glucose levels and KSHV lytic replication. High glucose levels induce oxidative stress and the production of H2O2, which mediates the reactivation of latent KSHV through multiple mechanisms. Our results provide the first experimental evidence and mechanistic support for the association of classic KS with diabetes.

HIV-1 Vpr Inhibits Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by Inducing MicroRNA miR-942-5p and Activating NF-κB Signaling.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is required for the development of several AIDS-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The high incidence of AIDS-KS has been ascribed to the interaction of KSHV and HIV-1. We have previously shown that HIV-1-secreted proteins Tat and Nef regulate the KSHV life cycle and synergize with KSHV oncogenes to promote angiogenesis and tumorigenesis. Here, we examined the regulation of KSHV latency by HIV-1 viral protein R (Vpr). We found that soluble Vpr inhibits the expression of KSHV lytic transcripts and proteins, as well as viral particle production by activating NF-κB signaling following internalization into PEL cells. By analyzing the expression profiles of microRNAs combined with target search by bioinformatics and luciferase reporter analyses, we identified a Vpr-upregulated cellular microRNA (miRNA), miR-942-5p, that directly targeted IκBα. Suppression of miR-942-5p relieved the expression of IκBα and reduced Vpr inhibition of KSHV lytic replication, while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV lytic replication. Our findings collectively illustrate that, by activating NF-κB signaling through upregulating a cellular miRNA to target IκBα, internalized HIV-1 Vpr inhibits KSHV lytic replication. These results have demonstrated an essential role of Vpr in the life cycle of KSHV. Coinfection by HIV-1 promotes the aggressive growth of Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). In this study, we have shown that soluble HIV-1 Vpr inhibits KSHV lytic replication by activating NF-κB signaling following internalization into PEL cells. Mechanistic studies revealed that a cellular microRNA upregulated by Vpr, miR-942-5p, directly targeted IκBα. Suppression of miR-942-5p relieved IκBα expression and reduced Vpr inhibition of KSHV replication, while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV replication. These results indicate that by activating NF-κB signaling through upregulating a cellular miRNA to target IκBα, internalized Vpr inhibits KSHV lytic replication. This work illustrates a molecular mechanism by which HIV-1-secreted regulatory protein Vpr regulates KSHV latency and the pathogenesis of AIDS-related malignancies.

Let-7 miRNA silencing promotes Kaposi's sarcoma-associated herpesvirus lytic replication via activating mitogen-activated protein kinase kinase kinase kinase 4 and its downstream factors

To explore the effect of let-7 miRNA silencing on Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and the underling mechanism. The pEGFP-C2-let-7 sponge vector was transfected into BCBL-1 and 293T cells with Lipofectamine 2000 to silence the expression of let-7 miRNAs. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of let-7 miRNAs, the transcriptional levels of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), cyclooxygenase-2 (COX-2) and matrix metalloproteinase 13 (MMP-13), and the DNA copy numbers of KSHV open reading frame 50 (ORF50) and open reading frame 72 (ORF72). Western blot was used to detect the total and phosphorylated protein levels of MAP4K4, COX-2, extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK. The expression of let-7 miRNAs was dramatically decreased in the let-7 sponge transfected BCBL-1 and 293T cells compared with that in the vector-transfected cells (P<0.05 for all). The gene copy number and mRNA transcriptional level of KSHV ORF50 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with that in the vector-transfected cells (1.00±0.10 vs. 2.33±0.18 and 1.08±0.48 vs 3.22±0.27, respectively,P<0.001 for both). The gene copy number and mRNA transcriptional level of KSHV ORF72 were also significantly increased in let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells(1.07±0.49 vs 1.67±0.45 and 1.01±0.19 vs 1.54±0.11, respectively,P<0.05 for both). Furthermore, the mRNA transcriptional levels of MAP4K4, COX-2 and MMP-13 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells (1.00±0.05 vs 5.73±0.96, 1.00±0.05 vs 2.68±0.19, 1.00±0.02 vs 2.69±0.25, respectively,P<0.001 for all). Let-7 miRNAs silencing also increased the protein expression levels of MAP4K4, COX-2 and phospho-ERK1/2, while the phospho-JNK and phospho-p38 were not changed in the BCBL-1 and 293T cells. Let-7 silencing may activate the replication of KSHV, possibly through up-regulating MAP4K4 and its downstream molecules COX-2, MMP-13, and phosphorylation of ERK1/2, finally results in the progression of Kaposi sarcoma.

Suppression of Kaposi's Sarcoma-Associated Herpesvirus Infection and Replication by 5'-AMP-Activated Protein Kinase.

The host intracellular antiviral restriction factors inhibit viral infection and replication. The 5'-AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. Activated AMPK inhibits the replication of numerous RNA viruses but enhances the entry of vaccinia virus. However, the role of AMPK in herpesvirus infection is unclear. In this study, we showed that the constitutive AMPK activity restricted Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in primary human umbilical vein endothelial cells while KSHV infection did not markedly affect the endogenous AMPK activity. Knockdown of the AMPKα1 considerably enhanced the expression of viral lytic genes and the production of infectious virions, while overexpression of a constitutively active AMPK had the opposite effects. Accordingly, an AMPK inhibitor, compound C, augmented viral lytic gene expressions and virion productions but an AMPK agonist, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), suppressed both. Furthermore, a common diabetes drug, metformin, which carries an AMPK-agonistic activity, drastically inhibited the expression of viral lytic genes and the production of infectious virions, suggesting the use of metformin as a therapeutic agent for KSHV infection and replication. Together, these results identify the host AMPK as a KSHV restriction factor that can serve as a potential therapeutic target. Host cells encode specific proteins to restrict viral infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. In this study, we have identified 5'-AMP-activated protein kinase (AMPK), a cellular energy sensor, as a restriction factor of KSHV lytic replication during primary infection. Activation of AMPK suppresses, while inhibition of AMPK enhances, KSHV lytic replication by regulating the expression of viral genes. AICAR and metformin, both of which are AMPK agonists currently used in clinics for the treatment of conditions associated with metabolic disorders, inhibit KSHV lytic replication. Thus, our work has identified AMPK as a potential therapeutic target and AICAR and metformin as potential therapeutic agents for KSHV-associated cancers.