Kaposi's Sarcoma-associated Herpesvirus Genome Research Articles

KSHV Rta Promoter Specification and Viral Reactivation

Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi’s sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic “CANT DNA repeats” in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta’s role as the switch is inefficient. Many factors modulate K-Rta’s function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation.

The Latency-Associated Nuclear Antigen, a Multifunctional Protein Central to Kaposi‘s Sarcoma-Associated Herpesvirus Latency

Latency-associated nuclear antigen (LANA) is encoded by the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) open reading frame 73. LANA is expressed during latent KSHV infection of cells, including tumor cells, such as primary effusion lymphoma, KS and multicentric Castleman's disease. Latently infected cells have multiple extrachromosomal copies of covalently closed circular KSHV genomes (episomes) that are stably maintained in proliferating cells. LANA's best characterized function is that of mediating episome persistence. It does so by binding terminal repeat sequences to the chromosomal matrix, thus ensuring episome replication with each cell division and efficient DNA segregation to daughter nuclei after mitosis. To achieve these functions, LANA associates with different host cell proteins, including chromatin-associated proteins and proteins involved in DNA replication. In addition to episome maintenance, LANA has transcriptional regulatory effects and affects cell growth. LANA exerts these functions through interactions with different cell proteins.

A Role for the Internal Repeat of the Kaposi's Sarcoma-Associated Herpesvirus Latent Nuclear Antigen in the Persistence of an Episomal Viral Genome

The latent nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the replication and partitioning of latent viral genomes. It contains an extended internal repeat (IR) region whose function is only incompletely understood. We constructed KSHV genomes lacking either LANA (KSHV-ΔLANA) or the IR region of LANA (KSHV-LANAΔ329-931). Although still capable of replicating a plasmid containing a latent origin of replication, LANAΔ329-931 does not support the establishment of stable cell lines containing a KSHV genome. These findings suggest a role for the LANA IR in KSHV episomal maintenance without its being required for replication.

Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication.

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences.

Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-κB inhibitory molecule (IκBα) and suppressed the transcriptional activity of NF-κB in PEL cells. The NF-κB specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-κB signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-κB signaling is upregulated by proteasome-dependent degradation of IκBα. The suppression of NF-κB signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.

Coordination of KSHV Latent and Lytic Gene Control by CTCF-Cohesin Mediated Chromosome Conformation

Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency control region is physically linked to the promoter regulatory region for ORF50, which encodes the KSHV immediate early protein RTA. Other linkages were also observed, including an interaction between the 5′ and 3′ end of the latency transcription cluster. Mutation of the CTCF-cohesin binding site reduced or eliminated the chromatin conformation linkages, and deregulated viral transcription and genome copy number control. siRNA depletion of CTCF or cohesin subunits also disrupted chromosomal linkages and deregulated viral latent and lytic gene transcription. Furthermore, the linkage between the latent and lytic control region was subject to cell cycle fluctuation and disrupted during lytic cycle reactivation, suggesting that these interactions are dynamic and regulatory. Our findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control.

Histone Demethylase JMJD2A Regulates Kaposi's Sarcoma-Associated Herpesvirus Replication and Is Targeted by a Viral Transcriptional Factor

The switch between the latency and lytic cycles of Kaposi's sarcoma-associated herpesvirus (KSHV) is accompanied by specific alterations of histone codes. Recently, comprehensive analysis of histone modifications of KSHV showed the deposition of H3K27me3 across the KSHV genome with two specific regions occupied by the heterochromatin marker H3K9me3. Here, we show that knockdown of JMJD2A, an H3K9me3 demethylase, attenuates viral titers, whereas its overexpression increases KSHV reactivation. JMJD2A is localized in regions of latent viral chromosomes that are deficient in the H3K9me3 mark, indicating that JMJD2A may be responsible for the low level of this mark on viral chromatin. The presence of JMJD2A on the latent genome maintains H3K9 in unmethylated form and signals the readiness of specific sets of viral genes to be reactivated. The demethylase activity of JMJD2A is important for KSHV reactivation, because a demethylase-deficient mutant cannot restore the JMJD2A knockdown phenotype. Interestingly, we found that the KSHV encoded K-bZIP associated with JMJD2A, resulting in the inhibition of demethylase activity of JMJD2A both in vivo and in vitro. Inhibition of JMJD2A by K-bZIP is likely due to a physical interaction which blocks substrate accessibility. A consequence of such an inhibition is increasing global levels of H3K9me3 and gene silencing. Consistently, K-bZIP overexpression resulted in a repression of ∼80% of the ≥2-fold differentially regulated genes compared to results for the uninduced control cells. The consequences of K-bZIP targeting JMJD2A during viral replication will be discussed. To our knowledge, this is the first description of a viral product shown to be a potent inhibitor of a host cellular histone demethylase.

Stability of a Long Noncoding Viral RNA Depends on a 9-Nt Core Element at the RNA 5' End to Interact with Viral ORF57 and Cellular PABPC1

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.

Functional characterization of Kaposi's sarcoma-associated herpesvirus open reading frame K8 by bacterial artificial chromosome-based mutagenesis.

The open reading frame K8 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a basic leucine zipper (bZip) protein that binds to the origin of viral DNA replication and is an integral component of viral lytic DNA replication complex. Moreover, K8 physically interacts with replication and transcription activator (RTA) and represses its transactivation activity on several viral promoters. To investigate the role of this protein in viral life cycle, we constructed two K8-null recombinant mutant viruses (BAC-ΔK8 and BAC-stopK8) by using a bacterial artificial chromosome (BAC) system. Latent viral infection can be reconstituted in 293T and BJAB cells with wild-type and the K8-null recombinant viruses by introducing the cloned viral genomes into the cells. When the cells carrying these viruses were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, no significant difference was seen in overall viral gene expression between wild-type and K8-null viruses, with lytic DNA replication still active in the latter. However, 293T cells harboring K8-null mutant viruses, either BAC-ΔK8 or BAC-stopK8, displayed lower copy numbers of latent KSHV genome in comparison with wild-type viruses. Furthermore, although K8 deficiency appeared to not affect infectivity when K8-null viruses were used to infect 293T, primary human microvascular dermal endothelial and human foreskin fibroblast cells, they exhibited much lower viral genome copy numbers in all types of cell compared to wild-type viruses. Taken together, these data suggest a possible role of K8 in abortive lytic DNA replication occurring in early stages of de novo infection or in the maintenance of latent viral genomes.

Characterization of entry and infection of monocytic THP-1 cells by Kaposi's sarcoma associated herpesvirus (KSHV): Role of heparan sulfate, DC-SIGN, integrins and signaling

KSHV effectively binds, enters and establishes infection in THP-1 cells with initial concurrent expression of latent ORF73 and lytic ORF50 genes and subsequent persistence of ORF73. KSHV genome persisted for 30 days and lytic cycle could be activated. KSHV utilized heparan sulfate for binding to THP-1 cells and primary monocytes. Blocking DC-SIGN did not inhibit KSHV binding; however, virus entry in THP-1 cells and in primary monocytes was reduced. In addition to the previously identified integrins α3β1, αvβ3 and αvβ5, integrin α5β1 was also utilized for infection. KSHV entered THP-1 cells via clathrin and caveolin mediated endocytosis and did not utilize macropinocytosis as in human dermal endothelial cells, and required an endosomal acidification. Infection also induced phosphorylation of FAK, Src, PI3K, NF-κB and ERK1/2 signaling molecules, and entry was blocked by tyrosine kinase inhibitors. These findings suggest that THP-1 cells are highly useful model for studying KSHV infection of monocytes.

Epigenetic Analysis of KSHV Latent and Lytic Genomes

Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle.

Bub1 and CENP-F Can Contribute to Kaposi's Sarcoma-Associated Herpesvirus Genome Persistence by Targeting LANA to Kinetochores

The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for segregation of viral episomes to progeny nuclei and allows for maintenance of the viral genome in newly divided daughter cells. LANA binds to KSHV terminal repeat (TR) DNA and simultaneously associates with chromatin-bound cellular proteins. This process tethers the viral episomes to host chromosomes. However, the mechanism of tethering is complex and involves multiple protein-protein interactions. Our previous proteomics studies which showed the association of LANA with centromeric protein F (CENP-F) prompted us to further study whether LANA targets centromeric proteins for persistence of KSHV episomes during cell division. Here we show that LANA colocalized with CENP-F as speckles, some of which are paired at centromeric regions of a subset of chromosomes in KSHV-infected JSC-1 cells. We also confirm that both the amino and carboxy termini of LANA can bind to CENP-F. Moreover, LANA associated with another kinetochore protein, Bub1 (budding uninhibited by benzimidazole 1), which is known to form a complex with CENP-F. Importantly, we demonstrated the dynamic association of LANA and Bub1/CENP-F and the colocalization between Bub1, LANA, and the KSHV episome tethered to the host chromosome using fluorescence in situ hybridization (FISH). Knockdown of Bub1 expression by lentivirus-delivered short hairpin RNA (shRNA) dramatically reduced the number of KSHV genome copies, whereas no dramatic effect was seen with CENP-F knockdown. Therefore, the interaction between LANA and the kinetochore proteins CENP-F and Bub1 is important for KSHV genome tethering and its segregation to new daughter cells, with Bub1 potentially playing a more critical role in the long-term persistence of the viral genome in the infected cell.

Small RNA profiling reveals antisense transcription throughout the KSHV genome and novel small RNAs

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus that encodes 12 precursor microRNAs (pre-miRNAs) that give rise to 17 different known approximately 22-nucleotide (nt) effector miRNAs. Like all herpesviruses, KSHV has two modes of infection: (1) a latent mode whereby only a subset of viral genes are expressed and (2) a lytic mode during which the full remaining viral genes are expressed. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. Here, we developed a method to profile small RNAs ( approximately 18-75 nt) from populations of cells undergoing predominantly lytic infection. Using two different next-generation sequencing platforms, we cloned and sequenced both pre-miRNAs and derivative miRNAs. Our analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5' end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. We report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miR-K12-8 and pre-miR-K12-12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. We identify several novel small RNAs of low abundance, including viral miRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. Finally, we observe widespread antisense transcription relative to known coding sequences during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, we observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs).

The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes

Herpesvirus latency is generally thought to be governed by epigenetic modifications, but the dynamics of viral chromatin at early timepoints of latent infection are poorly understood. Here, we report a comprehensive spatial and temporal analysis of DNA methylation and histone modifications during latent infection with Kaposi Sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi Sarcoma and primary effusion lymphoma (PEL). By use of high resolution tiling microarrays in conjunction with immunoprecipitation of methylated DNA (MeDIP) or modified histones (chromatin IP, ChIP), our study revealed highly distinct landscapes of epigenetic modifications associated with latent KSHV infection in several tumor-derived cell lines as well as de novo infected endothelial cells. We find that KSHV genomes are subject to profound methylation at CpG dinucleotides, leading to the establishment of characteristic global DNA methylation patterns. However, such patterns evolve slowly and thus are unlikely to control early latency. In contrast, we observed that latency-specific histone modification patterns were rapidly established upon a de novo infection. Our analysis furthermore demonstrates that such patterns are not characterized by the absence of activating histone modifications, as H3K9/K14-ac and H3K4-me3 marks were prominently detected at several loci, including the promoter of the lytic cycle transactivator Rta. While these regions were furthermore largely devoid of the constitutive heterochromatin marker H3K9-me3, we observed rapid and widespread deposition of H3K27-me3 across latent KSHV genomes, a bivalent modification which is able to repress transcription in spite of the simultaneous presence of activating marks. Our findings suggest that the modification patterns identified here induce a poised state of repression during viral latency, which can be rapidly reversed once the lytic cycle is induced.

Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1

Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). The far left-end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cellsUsing tandem affinity purification (TAP), we identified heat shock protein 90β (Hsp90β) and endoplasmic reticulum (ER)-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90β and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90α̣ isoform. We report that siRNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90 dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. Additionally, both Hsp90 and Hsp40/Erdj3 were essential for K1’s anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC50 values of 50nM and below.

Regulation of NF-κB inhibitor IκBα and viral replication by a KSHV microRNA

Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immune deficiency syndrome related malignancies including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman’s disease1. Control of viral lytic replication is essential for KSHV latency, evasion of host immune system, and induction of tumors1. Here, we show that deletion of a cluster of 14 microRNAs (miRs) from KSHV genome significantly enhances viral lytic replication as a result of reduced NF-κB activity. The miR cluster regulates NF-κB pathway by reducing the expression of IκBα protein, an inhibitor of the NF-κB complexes. Computational and miR seed mutagenesis analyses identify KSHV miR-K1 that directly mediates IκBα ?protein level by targeting the 3’UTR of its transcript. Expression of miR-K1 is sufficient to rescue the NF-κB activity and inhibit viral lytic replication while inhibition of miR-K1 in KSHV-infected PEL cells has the opposite effects. Thus, KSHV encodes a miR to control viral replication by activating NF-κB pathway. These results illustrate an important role for KSHV miRs in regulating viral latency and lytic replication by manipulating a host survival pathway.

Kaposi sarcoma-associated herpesvirus (KSHV): Molecular biology and oncogenesis

Kaposi sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA herpesvirus belonging to the γ-herpesvirinae subfamily. KSHV has been associated with the development of three neoplastic diseases: Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). In this review, we discuss the three KSHV-associated malignancies, KSHV genome, latent and lytic aspects of the viral lifecycle, putative viral oncogenes, as well as therapeutic regimens used for the treatment of KS, PEL, and MCD.

Genome-wide identification of binding sites for Kaposi's sarcoma-associated herpesvirus lytic switch protein, RTA

Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) encoded by ORF50 is a lytic switch protein for viral reactivation from latency. The expression of RTA activates the expression of downstream viral genes, and is necessary for triggering the full viral lytic program. Using chromatin immunoprecipitation assay coupled with a KSHV whole-genome tiling microarray (ChIP-on-chip) approach, we identified a set of 19 RTA binding sites in the KSHV genome in a KSHV-infected cell line BCBL-1. These binding sites are located in the regions of promoters, introns, or exons of KSHV genes including ORF8, ORFK4.1, ORFK5, PAN, ORF16, ORF29, ORF45, ORF50, ORFK8, ORFK10.1, ORF59, ORFK12, ORF71/72, ORFK14/ORF74, and ORFK15, the two origins of lytic replication OriLyt-L and OriLyt-R, and the microRNA cluster. We confirmed these RTA binding sites by ChIP and quantitative real-time PCR. We further mapped the RTA binding site in the first intron of the ORFK15 gene, and determined that it is RTA-responsive. The ORFK15 RTA binding sequence TTCCAGGAA TTCCTGGAA consists of a palindromic structure of two tandem repeats, of which each itself is also an imperfect inverted repeat. Reporter assay and electrophoretic mobility shift assay confirmed the binding of the RTA protein to this sequence in vitro. Sequence alignment with other RTA binding sites identified the RTA consensus binding motif as TTCCAGGAT(N) 0–16TTCCTGGGA. Interestingly, most of the identified RTA binding sites contain only half or part of this RTA binding motif. These results suggest the complexity of RTA binding in vivo, and the involvement of other cellular or viral transcription factors during RTA transactivation of target genes.

Treatment for Kaposi sarcoma herpesvirus: great challenges with promising accomplishments

Kaposi sarcoma-associated herpesvirus (KSHV) is associated with three distinct malignancies: Kaposi sarcoma (KS), primary effusion lymphoma, and multicentric Castleman disease. Treatment modalities for KSHV have largely been developed based on understanding its molecular pathogenesis. The KSHV genome has been fully sequenced and contains numerous genes with homology to human regulatory genes that are potentially important in KS development. Therapeutic strategies have been developed to target signaling pathways that are activated by KSHV. This review describes multiple classes of pharmacological compounds that have been studied in patients with KS, including antivirals, chemotherapeutics, and novel agents related to KSHV pathogenesis.

Accumulation of LANA at nuclear matrix fraction is important for Kaposi's sarcoma-associated herpesvirus replication in latency

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome replicates once per cell cycle, and the number of viral genome is maintained in the latency. The host cell-cycle-dependent replication of the viral genome is a fundamental process to critically keep the number of the genome. Here we show that the cellular pre-replication complex (pre-RC), the viral replication origin (ori-P) in a unit of the terminal repeat of the KSHV genome, and a viral replication factor, latency-associated nuclear antigen (LANA) accumulate at the nuclear matrix fraction in the G1 phase. We found not only that LANA itself was localized mainly to the nuclear matrix fraction but also that TR region of the KSHV genome existed together in the G1 phase. The localization of LANA at the nuclear matrix could be determined by structural consequence of the full length of LANA. Furthermore, transient replication assay revealed that the LANA's nuclear matrix localization was a pre-requisite for the efficient viral genome replication in the latency. Since LANA has been shown to bind the LANA binding sites (LBS) of the ori-P, these results suggest that LANA should recruit the ori-P to the nuclear matrix, where the complete pre-RC then forms on the ori-P, during the G1 phase. Thus, the nuclear matrix accumulation of cellular and viral replication factors is likely to be a key process for the initiation of replication of KSHV in the latency.