Kaposi's Sarcoma-associated Herpesvirus Lytic Reactivation Research Articles

Kaposi's sarcoma-associated herpesvirus microRNAs repress breakpoint cluster region protein expression, enhance Rac1 activity, and increase in vitro angiogenesis.

MicroRNAs (miRNAs) are small, ∼ 22-nucleotide-long RNAs that regulate gene expression posttranscriptionally. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs during latency, and the functional significance of these microRNAs during KSHV infection and their cellular targets have been emerging recently. Using a previously reported microarray profiling analysis, we identified breakpoint cluster region mRNA (Bcr) as a cellular target of the KSHV miRNA miR-K12-6-5p (miR-K6-5). Bcr protein levels were repressed in human umbilical vein endothelial cells (HUVECs) upon transfection with miR-K6-5 and during KSHV infection. Luciferase assays wherein the Bcr 3' untranslated region (UTR) was cloned downstream of a luciferase reporter showed repression in the presence of miR-K6-5, and mutation of one of the two predicted miR-K6-5 binding sites relieved this repression. Furthermore, inhibition or deletion of miR-K6-5 in KSHV-infected cells showed increased Bcr protein levels. Together, these results show that Bcr is a direct target of the KSHV miRNA miR-K6-5. To understand the functional significance of Bcr knockdown in the context of KSHV-associated disease, we hypothesized that the knockdown of Bcr, a negative regulator of Rac1, might enhance Rac1-mediated angiogenesis. We found that HUVECs transfected with miR-K6-5 had increased Rac1-GTP levels and tube formation compared to HUVECs transfected with control miRNAs. Knockdown of Bcr in latently KSHV-infected BCBL-1 cells increased the levels of viral RTA, suggesting that Bcr repression by KSHV might aid lytic reactivation. Together, our results reveal a new function for both KSHV miRNAs and Bcr in KSHV infection and suggest that KSHV miRNAs, in part, promote angiogenesis and lytic reactivation. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) infection is linked to multiple human cancers and lymphomas. KSHV encodes small nucleic acids (microRNAs) that can repress the expression of specific human genes, the biological functions of which are still emerging. This report uses a variety of approaches to show that a KSHV microRNA represses the expression of the human gene called breakpoint cluster region (Bcr). Repression of Bcr correlated with the activation of a protein previously shown to cause KS-like lesions in mice (Rac1), an increase in KS-associated phenotypes (tube formation in endothelial cells and vascular endothelial growth factor [VEGF] synthesis), and modification of the life cycle of the virus (lytic replication). Our results suggest that KSHV microRNAs suppress host proteins and contribute to KS-associated pathogenesis.

Activation of p90 ribosomal S6 kinases by ORF45 of Kaposi's sarcoma-associated herpesvirus is critical for optimal production of infectious viruses.

We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838-1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication. KSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

Inhibition of KAP1 Enhances Hypoxia-Induced Kaposi's Sarcoma-Associated Herpesvirus Reactivation through RBP-Jκ

Hypoxia-inducible factor 1α (HIF-1α) 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-1α 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-1α 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-Jκ and HIF-1α binding sites (RBS+HRE) within the same gene promoter and are tightly associated with KAP1. Inhibition of KAP1 greatly enhanced the association of RBP-Jκ with the HIF-1α 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-1α, 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-1α 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-Jκ (a key cellular regulator involved in Notch signaling) and HIF-1α binding sites. These sites are also tightly associated with KAP1. This discovery implies that KAP1, RBP-Jκ, and HIF-1α play an essential role in KSHV pathogenesis through subtle cross talk which is dependent on the oxygen levels in the infected cells.

Role of IRF4 in IFN-Stimulated Gene Induction and Maintenance of Kaposi Sarcoma–Associated Herpesvirus Latency in Primary Effusion Lymphoma Cells

IFN regulatory factor (IRF) 4 is a hematopoietic cell-specific transcription factor that regulates the maturation and differentiation of immune cells. Using an inducible expression system, we found that IRF4 directly induced a specific subset of IFN-stimulated genes (ISGs) in a type I IFN-independent manner in both epithelial and B cell lines. Moreover, Kaposi sarcoma-associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) enhances IRF4-mediated gene induction. Coexpression of IRF4 with vFLIP significantly increased ISG60 (IFIT3) and Cig5 (RSAD2) transcription that was dependent on the ability of vFLIP to activate NF-κB. A vFLIP mutant (A57L) defective in NF-κB activation failed to enhance IRF4-mediated ISG induction. Thus, we provide a physiologically relevant mechanism by which viral protein-mediated NF-κB activation modulates specific ISG induction by IRF4. In contrast, IRF4 also acted as a negative regulator of KSHV replication and transcription activator expression after induction of KSHV lytic reactivation in KSHV-positive primary effusion lymphoma cells. Taken together, these results suggest a dual role for IRF4 in regulating ISG induction and KSHV lytic reactivation in primary effusion lymphoma cells.

Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone

Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (∼5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.

Efficacy of bortezomib in a direct xenograft model of primary effusion lymphoma

Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma most commonly diagnosed in HIV-positive patients and universally associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Chemotherapy treatment of PEL yields only short-term remissions in the vast majority of patients, but efforts to develop superior therapeutic approaches have been impeded by lack of animal models that accurately mimic human disease. To address this issue, we developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells. We used this model to show that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions. The proapoptotic effects of bortezomib are not mediated by inhibition of the prosurvival NF-kappaB pathway or by induction of a terminal unfolded protein response. Transcriptome analysis by genomic arrays revealed that bortezomib down-regulated cell-cycle progression, DNA replication, and Myc-target genes. Furthermore, we demonstrate that in vivo treatment with either bortezomib or doxorubicin induces KSHV lytic reactivation. These reactivations were temporally distinct, and this difference may help elucidate the therapeutic window for use of antivirals concurrently with chemotherapy. Our findings show that this direct xenograft model can be used for testing novel PEL therapeutic strategies and also can provide a rational basis for evaluation of bortezomib in clinical trials.

Enhancement of Autophagy during Lytic Replication by the Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Activator

Autophagy is one of two major degradation systems in eukaryotic cells. The degradation mechanism of autophagy is required to maintain the balance between the biosynthetic and catabolic processes and also contributes to defense against invading pathogens. Recent studies suggest that a number of viruses can evade or subvert the host cell autophagic pathway to enhance their own replication. Here, we investigated the effect of autophagy on the KSHV (Kaposi's sarcoma-associated herpesvirus) life cycle. We found that the inhibition of autophagy reduces KSHV lytic reactivation from latency, and an enhancement of autophagy can be detected during KSHV lytic replication. In addition, RTA (replication and transcription activator), an essential viral protein for KSHV lytic reactivation, is able to enhance the autophagic process, leading to an increase in the number of autophagic vacuoles, an increase in the level of the lipidated LC3 protein, and the formation of autolysosomes. Moreover, the inhibition of autophagy affects RTA-mediated lytic gene expression and viral DNA replication. These results suggest that RTA increases autophagy activation to facilitate KSHV lytic replication. This is the first report demonstrating that autophagy is involved in the lytic reactivation of KSHV.

Activation of RSK and ERK MAPK kinases by ORF45 of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus etiologically linked to Kaposi's sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman's disease. Infection and reactivation of KSHV activates multiple MAPK pathways. Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency, but the responsible viral factors and underlying mechanism was unknown. Open reading frame 45 (ORF45) of KSHV is an immediate early, phosphorylated, and tegument protein. Its unique temporal and spatial expression put it in the forefront of coping with host cellular environment. We recently reported that ORF45 interacts with p90 ribosomal S6 kinases (RSKs), a family of serine/threonine kinases that lie at the terminus of the ERK pathway, and strongly stimulates their kinase activities. We found that binding of ORF45 to RSK increases the association of ERK with RSK, such that ORF45, RSK, and ERK form complexes. The complexes shield active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK are activated and sustained at high levels. We also demonstrated that RSK and ERK are activated biphasically during KSHV primary infection and lytic replication cycle. We provided evidence that the reciprocal activation of ERK and RSK by ORF45 contributes to the sustained activation of ERK/RSK in KSHV lytic replication. We further demonstrated that ablation of RSK expression by SiRNA or inhibition of kinase activity by specific RSK inhibitors lead to lower KSHV lytic gene expression, reactivation, and virus production, suggesting an essential role of the RSK in KSHV lytic replication. Therefore, inhibition of RSK is likely to disrupt KSHV infection and be a potent target for therapy of KSHV associated diseases.

Effects of NFκB activation on KSHV latency and lytic reactivation are complex and context-dependent

Like all herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) can produce either latent or lytic infection. The latent v-FLIP gene is a strong activator of NFκB, and in primary effusion lymphoma (PEL) cells, blockade of NFκB activation is associated with enhanced lytic gene expression, while overexpression of p65 impairs expression of reporter genes driven by lytic promoters. This has led to the suggestion that NFκB activation may promote latency by suppressing lytic reactivation. Here we examine in detail the effects of NFκB activation on KSHV replication in several cell types. In accord with earlier work, we find that inhibition of NFκB signaling in PEL cells is associated with enhanced lytic reactivation of KSHV. Similarly, in de novo KSHV infection of primary endothelial cells, inhibition of NFκB signaling leads to an increase in lytic gene expression and enhanced virion production. By contrast, KSHV-infected human foreskin fibroblasts (HFF) show no increase in spontaneous lytic reactivation when NFκB is inhibited. Moreover, if NFκB activation is always inhibitory to lytic gene expression, one might expect its activation to be suppressed during the lytic cycle. However, we find that NFκB signaling is strongly and consistently activated in lytically infected cells of all lineages. Together these data indicate that (i) the relationship of NFκB activation to latency and lytic reactivation is not uniform, but is dependent on the cellular context; and (ii) even though NFκB activation is inhibitory to lytic gene expression in some contexts, such inhibition is at least partially bypassed or overridden during lytic growth.

Antiviral effect of Alloferon on Kaposi's sarcoma‐associated herpesvirus

Kaposi's sarcoma‐associated herpesvirus (KSHV) is a human gammaherpesvirus that is associated with the development of Kaposi's sarcoma and primary effusion lymphoma. KSHV establishes two alternate genetic life cycle programs upon infection of its host cells, which maintain a latent infection in infected cells but undergo lytic reactivation in response to various stimuli. In this study, we used BCBL‐1, persistent KSHV‐infected B lymphoma cell line, where the lytic and latent cycle of KSHV was regulated by TPA treatment. Alloferon is an immunomodulating peptide originating from insects which has been successfully developed in clinical trials as antiviral drug in Russia and showed remarkable effect on prevention of recurrent HSV infection. However, its specific mechanism remains to be clarified. We investigated the effect of alloferon on the regulation of lytic reactivation in viral infected cells. We found that alloferon effectively suppressed the down‐regulation of cell proliferation induced by TPA treatment. It suggests that the lytic reactivation of KSHV within BCBL‐1 can be regulated by alloferon. We next investigated the specific intracellular target of alloferon. Alloferon regulated the expression level of RTA as lytic switch protein, and that of ORF45 and vIRF2, which inhibit the production of type I IFNs. Therefore, we suggests that alloferon may be an effective antiviral agent on KSHV infection.

X box binding protein XBP-1s transactivates the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF50 promoter, linking plasma cell differentiation to KSHV reactivation from latency.

Reactivation of lytic replication from viral latency is a defining property of all herpesviruses. Despite this, the authentic physiological cues for the latent-lytic switch are unclear. Such cues should ensure that viral lytic replication occurs under physiological conditions, predominantly in sites which facilitate transmission to permissive uninfected cells and new susceptible hosts. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with the B-cell neoplasm primary effusion lymphoma (PEL), in which the virus remains latent. We have previously shown that PEL cells have the gene expression profile and immunophenotype of cycling preplasma cells (plasmablasts). Here, we show that the highly active spliced isoform of plasma cell transcription factor X box binding protein 1 (XBP-1s) is a lytic switch for KSHV. XBP-1s is normally absent in PEL, but the induction of endoplasmic reticulum stress leads to XBP-1s generation, plasma cell-like differentiation, and lytic reactivation of KSHV. XBP-1s binds to and activates the KSHV immediate-early gene ORF50 and synergizes with the ORF50 gene product RTA to induce a full lytic cycle. These data suggest that KSHV remains latent until B-cell terminal differentiation into plasma cells, the transcriptional environment of which provides the physiological "lytic switch" through XBP-1s. This links B-cell terminal differentiation to KSHV lytic reactivation.

Highly selective escape from KSHV-mediated host mRNA shutoff and its implications for viral pathogenesis.

During Kaposi's sarcoma (KS)–associated herpesvirus (KSHV) lytic infection, many virus-encoded signaling molecules (e.g., viral G protein–coupled receptor [vGPCR]) are produced that can induce host gene expression in transiently transfected cells, and roles for such induced host genes have been posited in KS pathogenesis. However, we have recently found that host gene expression is strongly inhibited by 10–12 h after lytic reactivation of KSHV, raising the question of whether and to what extent de novo host gene expression induced by viral signaling molecules can proceed during the lytic cycle. Here, we show by microarray analysis that expression of most vGPCR target genes is drastically curtailed by this host shutoff. However, rare cellular genes can escape the host shutoff and are potently up-regulated during lytic KSHV growth. Prominent among these is human interleukin-6, whose striking induction may contribute to the overexpression of this cytokine in several disease states linked to KSHV infection.

Suppression of tetradecanoyl phorbol acetate-induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus by K1 signal transduction.

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region and elicits cellular signal transduction through this motif. To investigate the role of K1 signal transduction in KSHV replication, we expressed full-length K1 and CD8-K1 chimeras in BCBL1 cells. Unlike its strong signaling activity in uninfected B lymphocytes, K1 did not induce intracellular calcium mobilization or NF-AT activation at detectable levels in KSHV-infected BCBL1 cells. Instead, K1 signaling dramatically suppressed KSHV lytic reactivation induced by tetradecanoyl phorbol acetate (TPA) stimulation, but not by ORF50 ectopic expression. Mutational analysis showed that the cytoplasmic ITAM sequence of K1 was required for this suppression. Viral microarray and immunoblot analyses demonstrated that K1 signaling suppressed the TPA-mediated increase in the expression of a large subset of viral lytic genes in KSHV-infected BCBL1 cells. Furthermore, electrophoretic mobility shift assays demonstrated that TPA-induced activation of AP-1, NF-kappaB, and Oct-1 activities was severely diminished in BCBL1 cells expressing the K1 cytoplasmic domain. The reduced activities of these transcription factors may confer the observed reduction in viral lytic gene expression. These results demonstrate that K1-mediated signal transduction in KSHV-infected cells is profoundly different from that in KSHV-negative cells. Furthermore, K1 signal transduction efficiently suppresses TPA-mediated viral reactivation in an ITAM-dependent manner, and this suppression may contribute to the establishment and/or maintenance of KSHV latency in vivo.