Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus interacts with origin recognition complexes at the LANA binding sequence within the terminal repeats

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the cis-acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G(1)/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.     

ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin

The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.     

Mitotic chromosome-binding activity of latency-associated nuclear antigen 1 is required for DNA replication from terminal repeat sequence of Kaposi's sarcoma-associated herpesvirus

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the persistence of the viral genome during latent infection. It has been suggested that LANA1 tethers the viral genome to the host chromosome and also participates actively in DNA replication from the terminal repeat of KSHV. Here we show by mutational analysis that the mitotic chromosome-binding activity of LANA1 is tightly coupled to its replication activity. Thus, KSHV appears to have evolved a unique tactic for its stable maintenance.     

Poly(ADP-ribose) polymerase 1 binds to Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat sequence and modulates KSHV replication in latency

During latency, Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to replicate once and to be partitioned in synchrony with the cell cycle of the host. In this replication cycle, the KSHV terminal repeat (TR) sequence functions as a replication origin, assisted by the latency-associated nuclear antigen (LANA). Thus, TR seems to function as a cis element for the replication and partitioning of the KSHV genome. Viral replication and partitioning are also likely to require cellular factors that interact with TR in either a LANA-dependent or -independent manner. Here, we sought to identify factors that associate with TR by using a TR DNA column and found that poly(ADP-ribose) polymerase 1 (PARP1) and known replication factors, including ORC2, CDC6, and Mcm7, bound to TR. PARP1 bound directly to a specific region within TR independent of LANA, and LANA was poly(ADP-ribosyl)ated by PARP1. Drugs such as hydroxyurea and niacinamide, which raise or lower PARP activity, respectively, affected the virus copy number in infected cells. Thus, the poly(ADP-ribosyl)ation status of LANA appears to affect the replication and/or maintenance of the viral genome. Drugs that specifically up-regulate PARP activity may lead to the disappearance of latent KSHV.     

ORF73 of herpesvirus Saimiri strain C488 tethers the viral genome to metaphase chromosomes and binds to cis-acting DNA sequences in the terminal repeats

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a human oncogenic gamma-2-herpesvirus, transforms human endothelial cells and establishes latent infection at a low efficiency in vitro. During latent infection, only a limited number of genes are expressed, and the circularized viral genome is maintained as a multicopy episome. Latency-associated nuclear antigen (LANA), exclusively expressed during latency, has been shown to have a multifunctional role in KS pathogenesis. LANA tethers the viral episome to the host chromosome, thus ensuring efficient persistence of the viral genome during successive rounds of cell division. Besides episome maintenance, LANA modulates the expression of genes of various cellular and viral pathways, including those of retinoblastoma protein and p53. Herpesvirus saimiri (HVS), another gamma-2-herpesvirus, primarily infects New World primates. Orf73, encoding the nuclear antigen of HVS, is the positional homolog of the LANA gene, and the ORF73 protein has some sequence homology to KSHV LANA. However, the function of ORF73 of HVS has not been thoroughly investigated. In this report, we show that HVS ORF73 may be important for episome persistence and colocalizes with the HVS genomic DNA on metaphase chromosomes. Furthermore, HVS terminal repeats (TRs) contain a cis-acting sequence similar to that in KSHV TRs, suggesting that the LANA binding sequence is conserved between these two viruses. This cis-acting element is sufficient to bind HVS ORF73 from strains C488 and A11, and plasmids containing the HVS C488 TR element are maintained and replicate in HVS C488 ORF73-expressing cells.     

Kaposi's sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen inhibits lytic replication by targeting Rta: a potential mechanism for virus-mediated control of latency

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.     

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.     

DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi’s sarcoma-associated herpesvirus latent replication

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/−) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.     

The ubiquitin-specific protease USP7 modulates the replication of Kaposi's sarcoma-associated herpesvirus latent episomal DNA

Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987-29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins.     

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.     

The minimal replicator element of the Kaposi's sarcoma-associated herpesvirus terminal repeat supports replication in a semiconservative and cell-cycle-dependent manner

Kaposi's sarcoma-associated herpesvirus (KSHV) persists as episomes in infected cells by circularizing at the terminal repeats (TRs). The KSHV episome carries multiple reiterated copies of the terminal repeat, and each copy is capable of supporting replication. Expression of the latency-associated nuclear antigen (LANA) is critical for the replication of TR-containing plasmids. A 32-bp sequence upstream of LANA binding site 1 (LBS1), referred to as RE (replication element), along with LANA binding sites 1 and 2 (RE-LBS1/2), is sufficient to support replication (J. Hu and R. Renne, J. Virol. 79:2637-2642, 2005). In this report we demonstrate that the minimal replicator element (RE-LBS1/2) replicates in synchrony with the host cellular DNA, and only once, in a cell-cycle-dependent manner. Overexpression of the mammalian replication inhibitor geminin blocked replication of the plasmid containing the minimal replicator element, confirming the involvement of the host cellular replication control mechanism, and prevented rereplication of the plasmid in the same cell cycle. Overexpression of Cdt1 also rescued the replicative ability of the RE-LBS1/2-containing plasmids. A chromatin immunoprecipitation assay performed using anti-origin recognition complex 2 (alpha-ORC2) and alpha-LANA antibodies from cells transfected with RE-LBS1/2, RE-LBS1, LBS1, or RE showed the association of ORC2 with the RE region. Expression of LANA increased the number of copies of chromatin-bound DNA of replication elements, suggesting that LANA is important for the recruitment of ORCs and may contribute to the stabilization of the replication protein complexes at the RE site.     

Chromosome binding site of latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus is essential for persistent episome maintenance and is functionally replaced by histone H1

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (DeltaN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of DeltaN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.     

LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection

One of the hallmarks of the latent phase of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection is the global repression of lytic viral gene expression. Following de novo KSHV infection, the establishment of latency involves the chromatinization of the incoming viral genomes and recruitment of the host Polycomb repressive complexes (PRC1 and PRC2) to the promoters of lytic genes, which is accompanied by the inhibition of lytic genes. However, the mechanism of how PRCs are recruited to the KSHV episome is still unknown. Utilizing a genetic screen of latent genes in the context of KSHV genome, we identified the latency-associated nuclear antigen (LANA) to be responsible for the genome-wide recruitment of PRCs onto the lytic promoters following infection. We found that LANA initially bound to the KSHV genome right after infection and subsequently recruited PRCs onto the viral lytic promoters, thereby repressing lytic gene expression. Furthermore, both the DNA and chromatin binding activities of LANA were required for the binding of LANA to the KSHV promoters, which was necessary for the recruitment of PRC2 to the lytic promoters during de novo KSHV infection. Consequently, the LANA-knockout KSHV could not recruit PRCs to its viral genome upon de novo infection, resulting in aberrant lytic gene expression and dysregulation of expression of host genes involved in cell cycle and proliferation pathways. In this report, we demonstrate that KSHV LANA recruits host PRCs onto the lytic promoters to suppress lytic gene expression following de novo infection.     

An autonomous replicating element within the KSHV genome

Members of the herpesviridae family including Kaposi's sarcoma-associated herpesvirus (KSHV) persist latently in their hosts and harbor their genomes as closed circular episomes. Propagation of the KSHV genome into new daughter cells requires replication of the episome once every cell division and is considered critically dependent on expression of the virus encoded latency-associated nuclear antigen (LANA). This study demonstrates a LANA-independent mechanism of KSHV latent DNA replication. A cis-acting DNA element within a discreet KSHV genomic region termed the long unique region (LUR) can initiate and support replication of plasmids lacking LANA-binding sequences or a eukaryotic replication origin. The human cellular replication machinery proteins ORC2 and MCM3 associated with the LUR element and depletion of cellular ORC2 abolished replication of the plasmids indicating that recruitment of the host cellular replication machinery is important for LUR-dependent replication. Thus, KSHV can initiate replication of its genome independent of any trans-acting viral factors.     

Molecular biology and pathogenesis of Kaposi sarcoma-associated herpesvirus

Kaposi sarcoma (KS)-associated herpesvirus (KSHV) is the most recently discovered human oncogenic herpesvirus. The virus is associated with KS lesions and other human malignancies, including pleural effusion lymphomas and multicentric castleman's disease. The sequence of the viral genome demonstrated that it belongs to the gammaherpesvirus family similar to the Epstein-Barr virus, the only other known human herpesvirus associated with human cancers. Molecular studies have identified a number of viral genes involved in regulation of cell proliferation, gene regulation, chromatin remodeling and apoptosis. KSHV transforms human endothelial cells in vitro with low efficiency and expresses a repertoire of latent genes involved in the establishment of latency. One of these latent proteins, the latency-associated nuclear antigen (LANA) is required for episomal maintenance and tethers the viral genome to the host chromatin. LANA has now been shown to be a multifunctional protein involved in numerous cellular functions including binding to the retinoblastoma protein and p53, regulating cell proliferation and apoptosis.