Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers.
ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.
Project description:Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.
Project description:Hypoxia-inducible factor 1? (HIF-1?) has been frequently implicated in many cancers as well as viral pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several human malignancies. It can stabilize HIF-1? during latent infection and undergoes lytic replication in response to hypoxic stress. However, the mechanism by which KSHV controls its latent and lytic life cycle through the deregulation of HIF-1? is not fully understood. Our previous studies showed that the hypoxia-sensitive chromatin remodeler KAP1 was targeted by the KSHV-encoded latency-associated nuclear antigen (LANA) to repress expression of the major lytic replication and transcriptional activator (RTA). Here we further report that an RNA interference-based knockdown of KAP1 in KSHV-infected primary effusion lymphoma (PEL) cells disrupted viral episome stability and abrogated sub-G1/G1 arrest of the cell cycle while increasing the efficiency of KSHV lytic reactivation by hypoxia or using the chemical 12-O-tetradecanoylphorbol-13-acetate (TPA) or sodium butyrate (NaB). Moreover, KSHV genome-wide screening revealed that four hypoxia-responsive clusters have a high concurrence of both RBP-J? and HIF-1? binding sites (RBS+HRE) within the same gene promoter and are tightly associated with KAP1. Inhibition of KAP1 greatly enhanced the association of RBP-J? with the HIF-1? complex for driving RTA expression not only in normoxia but also in hypoxia. These results suggest that both KAP1 and the concurrence of RBS+HRE within the RTA promoter are essential for KSHV latency and hypoxia-induced lytic reactivation.Kaposi's sarcoma-associated herpesvirus (KSHV), a DNA tumor virus, is an etiological agent linked to several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). HIF-1?, a key hypoxia-inducible factor, is frequently elevated in KSHV latently infected tumor cells and contributes to KSHV lytic replication in hypoxia. The molecular mechanisms of how KSHV controls the latent and lytic life cycle through deregulating HIF-1? remain unclear. In this study, we found that inhibition of hypoxia-sensitive chromatin remodeler KAP1 in KSHV-infected PEL cells leads to a loss of viral genome and increases its sensitivity to hypoxic stress, leading to KSHV lytic reactivation. Importantly, we also found that four hypoxia-responsive clusters within the KSHV genome contain a high concurrence of RBP-J? (a key cellular regulator involved in Notch signaling) and HIF-1? binding sites. These sites are also tightly associated with KAP1. This discovery implies that KAP1, RBP-J?, and HIF-1? play an essential role in KSHV pathogenesis through subtle cross talk which is dependent on the oxygen levels in the infected cells.
Project description:The majority of AIDS-associated primary effusion lymphomas (PEL) are latently infected with both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). PELs harboring two viruses have higher oncogenic potential, suggesting functional interactions between EBV and KSHV. The KSHV replication and transcription activator (K-RTA) is necessary and sufficient for induction of KSHV lytic replication. EBV latent membrane protein 1 (LMP-1) is essential for EBV transformation and establishment of latency in vitro. We show EBV inhibits chemically induced KSHV lytic replication, in part because of a regulatory loop in which K-RTA induces EBV LMP-1 and LMP-1 in turn inhibits K-RTA expression and furthermore the lytic gene expression of KSHV. Suppression of LMP-1 expression in dually infected PEL cells enhances the expression of K-RTA and lytic replication of KSHV upon chemical induction. Because LMP-1 is known to inhibit EBV lytic replication, KSHV-mediated induction of LMP-1 would potentiate EBV latency. Moreover, KSHV infection of EBV latency cells induces LMP-1, and K-RTA is involved in the induction. Both LMP-1 and K-RTA are expressed during primary infection by EBV of KSHV latency cells. Our findings provide evidence that an interaction between EBV and KSHV at molecular levels promotes the maintenance and possibly establishment of viral latency, which may contribute to pathogenesis of PELs.
Project description:Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.
Project description: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.
Project description:The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human proliferative disorders, namely, Kaposi's sarcoma, primary effusion lymphomas (PEL), and multicentric Castleman's disease. Lytic DNA replication of KSHV, which is essential for viral propagation, requires the binding of at least two KSHV proteins, replication and transactivation activator (RTA) and K-bZIP, on the lytic origin of replication. Moreover, K-bZIP physically interacts with RTA and represses its transactivation activity on several viral promoters in transient transfection assays. To evaluate the physiological roles of K-bZIP in the context of PEL, we generated BCBL-1 cells with a tetracycline (Tet)-inducible small hairpin RNA (shRNA) directed against the K8 mRNA to knock down K-bZIP expression at different points during KSHV's life cycle. Using this model, we demonstrate that in the absence of K-bZIP expression, dramatic decreases in orf50, orf57, and orf26 transcript expression are observed. Similar effects were seen at the protein level for RTA (immediate-early protein) and K8.1 (late protein) expression. Interestingly, a direct correlation between K-bZIP levels and viral lytic mRNAs was noticed. As a consequence of K-bZIP knockdown, viral DNA replication and virion production were severely impaired. The same effects were observed following knockdown of K-bZIP in another PEL cell line, BC3. Finally, using shRNA-K8-inducible 293 cells, we report that de novo synthesis of K-bZIP is not necessary for initiation of infection and latency establishment. These data support the concept that K-bZIP is essential for lytic viral gene expression, viral DNA replication, and virus propagation in PEL cells.
Project description:Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.
Project description:Noncoding RNAs have substantial effects in host-virus interactions. Circular RNAs (circRNAs) are novel single-stranded noncoding RNAs which can decoy other RNAs or RNA-binding proteins to inhibit their functions. The role of circRNAs is largely unknown in the context of Kaposi's sarcoma herpesvirus (KSHV). We hypothesized that circRNAs influence viral infection by inhibiting host and/or viral factors. Transcriptome analysis of KSHV-infected primary endothelial cells and a B cell line identified human circRNAs that are differentially regulated upon infection. We confirmed the expression changes with divergent PCR primers and RNase R treatment of specific circRNAs. Ectopic expression of hsa_circ_0001400, a circRNA induced by infection, suppressed expression of key viral latent gene LANA and lytic gene RTA in KSHV de novo infections. Since human herpesviruses express noncoding RNAs like microRNAs, we searched for viral circRNAs encoded in the KSHV genome. We performed circRNA-Seq analysis with RNase R-treated, circRNA-enriched RNA from KSHV-infected cells. We identified multiple circRNAs encoded by the KSHV genome that are expressed in KSHV-infected endothelial cells and primary effusion lymphoma (PEL) cells. The KSHV circRNAs are located within ORFs of viral lytic genes, are up-regulated upon the induction of the lytic cycle, and alter cell growth. Viral circRNAs were also detected in lymph nodes from patients of KSHV-driven diseases such as PEL, Kaposi's sarcoma, and multicentric Castleman's disease. We revealed new host-virus interactions of circRNAs: human antiviral circRNAs are activated in response to KSHV infection, and viral circRNA expression is induced in the lytic phase of infection.