Latency-associated Nuclear Antigen Binding Sites Research Articles

The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Inhibits Expression of SUMO/Sentrin-Specific Peptidase 6 To Facilitate Establishment of Latency.

Kaposi's sarcoma-associated herpesvirus (KSHV), which belongs to the Gammaherpesviridae, typically displays two different phases in its life cycle, the latent phase and the lytic phase. Latency-associated nuclear antigen (LANA), the primary viral product during latency, has been reported to bind to a series of cellular gene promoters to modulate gene transcription. To systemically elucidate the cellular genes regulated by LANA, we identified genome-wide LANA binding sites by chromatin immunoprecipitation coupled with sequencing (ChIP-seq). We stratified ChIP-seq data and found that LANA might be involved in the macromolecule catabolic process. Specifically, we found and verified that LANA could directly bind to the promoter of the SUMO/sentrin-specific peptidase 6 (SENP6) gene in vivo and in vitro LANA could repress SENP6 promoter activity in a dose-dependent manner in a reporter gene assay. LANA expression was sufficient to inhibit endogenous SENP6 expression at both the RNA and protein levels. Moreover, SENP6 overexpression in KSHV-infected cells reduced LANA at the protein level. Mechanistically, we found that SENP6 could interact with LANA and reduce the formation of sumoylated LANA, which relies on the desumoylation ability of SENP6. During de novo infection, SENP6 overexpression would decrease the abundance of LANA and enhance viral gene expression, which would hamper the establishment of latency. Taken together, these data suggest that KSHV-encoded LANA could inhibit SENP6 expression to regulate the abundance of itself, which may play an important role in controlling the establishment of latency.IMPORTANCE LANA, as a key latent protein produced by KSHV, is responsible for episome persistence and regulates viral reactivation. In the present study, our results demonstrated that LANA could bind to the promoter region of the SENP6 gene and inhibit SENP6 expression while the regulated SENP6 could in turn modulate the abundance of LANA through desumoylation. This delicate regulation may provide important insights to explain the abundance of LANA during KSHV latency.

Epigenetic Landscape of Kaposi's Sarcoma-Associated Herpesvirus Genome in Classic Kaposi's Sarcoma Tissues

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically related to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). It typically displays two different phases in its life cycle, the default latency and occasional lytic replication. The epigenetic modifications are thought to determine the fate of KSHV infection. Previous studies elegantly depicted epigenetic landscape of latent viral genome in in vitro cell culture systems. However, the physiologically relevant scenario in clinical KS tissue samples is unclear. In the present study, we established a protocol of ChIP-Seq for clinical KS tissue samples and mapped out the epigenetic landscape of KSHV genome in classic KS tissues. We examined AcH3 and H3K27me3 histone modifications on KSHV genome, as well as the genome-wide binding sites of latency associated nuclear antigen (LANA). Our results demonstrated that the enriched AcH3 was mainly restricted at latent locus while H3K27me3 was widespread on KSHV genome in classic KS tissues. The epigenetic landscape at the region of vIRF3 gene confirmed its silenced state in KS tissues. Meanwhile, the abundant enrichment of LANA at the terminal repeat (TR) region was also validated in the classic KS tissues, however, different LANA binding sites were observed on the host genome. Furthermore, we verified the histone modifications by ChIP-qPCR and found the dominant repressive H3K27me3 at the promoter region of replication and transcription activator (RTA) in classic KS tissues. Intriguingly, we found that the TR region in classic KS tissues was lacking in AcH3 histone modifications. These data now established the epigenetic landscape of KSHV genome in classic KS tissues, which provides new insights for understanding KSHV epigenetics and pathogenesis.

KSHV Genome Replication and Maintenance.

Kaposi's sarcoma associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is a major etiological agent for multiple severe malignancies in immune-compromised patients. KSHV establishes lifetime persistence in the infected individuals and displays two distinct life cycles, generally a prolonged passive latent, and a short productive or lytic cycle. During latent phase, the viral episome is tethered to the host chromosome and replicates once during every cell division. Latency-associated nuclear antigen (LANA) is a predominant multifunctional nuclear protein expressed during latency, which plays a central role in episome tethering, replication and perpetual segregation of the episomes during cell division. LANA binds cooperatively to LANA binding sites (LBS) within the terminal repeat (TR) region of the viral episome as well as to the cellular nucleosomal proteins to tether viral episome to the host chromosome. LANA has been shown to modulate multiple cellular signaling pathways and recruits various cellular proteins such as chromatin modifying enzymes, replication factors, transcription factors, and cellular mitotic framework to maintain a successful latent infection. Although, many other regions within the KSHV genome can initiate replication, KSHV TR is important for latent DNA replication and possible segregation of the replicated episomes. Binding of LANA to LBS favors the recruitment of various replication factors to initiate LANA dependent DNA replication. In this review, we discuss the molecular mechanisms relevant to KSHV genome replication, segregation, and maintenance of latency.

KSHV but not MHV-68 LANA induces a strong bend upon binding to terminal repeat viral DNA.

Latency-associated nuclear antigen (LANA) is central to episomal tethering, replication and transcriptional regulation of γ2-herpesviruses. LANA binds cooperatively to the terminal repeat (TR) region of the viral episome via adjacent LANA binding sites (LBS), but the molecular mechanism by which LANA assembles on the TR remains elusive. We show that KSHV LANA and MHV-68 LANA proteins bind LBS DNA using strikingly different modes. Solution structure of LANA complexes revealed that while kLANA tetramer is intrinsically bent both in the free and bound state to LBS1–2 DNA, mLANA oligomers instead adopt a rigid linear conformation. In addition, we report a novel non-ring kLANA structure that displays more flexibility at its assembly interface than previously demonstrated. We identified a hydrophobic pivot point located at the dimer–dimer assembly interface, which gives rotational freedom for kLANA to adopt variable conformations to accommodate both LBS1–2 and LBS2–1–3 DNA. Alterations in the arrangement of LBS within TR or at the tetramer assembly interface have a drastic effect on the ability of kLANA binding. We also show kLANA and mLANA DNA binding functions can be reciprocated. Although KSHV and MHV-68 are closely related, the findings provide new insights into how the structure, oligomerization, and DNA binding of LANA have evolved differently to assemble on the TR DNA.

The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA

Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains ("LANA speckles"), a hallmark of KSHV latency.

LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after de novo infection, (ii) interacts with transcriptional regulators and chromatin remodelers, and (iii) regulates the LANA and RTA promoters, we hypothesized that LANA may contribute to the establishment of latency through epigenetic control. We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.

Protein Arginine Methyltransferase 1-directed Methylation of Kaposi Sarcoma-associated Herpesvirus Latency-associated Nuclear Antigen

The Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a multifunctional protein with roles in gene regulation and maintenance of viral latency. Post-translational modification of LANA is important for functional diversification. Here, we report that LANA is subject to arginine methylation by protein arginine methyltransferase 1 in vitro and in vivo. The major arginine methylation site in LANA was mapped to arginine 20. This site was mutated to either phenylalanine (bulky hydrophobic, constitutive methylated mimetic) or lysine (positively charged, non-arginine methylatable) residues. The significance of the methylation in LANA function was examined in both the isolated form and in the context of the viral genome through the generation of recombinant KSHV. In addition, authentic LANA binding sites on the KSHV episome in naturally infected cells were identified using a whole genome KSHV tiling array. Although mutation of the methylation site resulted in no significant difference in KSHV LANA subcellular localization, we found that the methylation mimetic mutation resulted in augmented histone binding in vitro and increased LANA occupancy at identified LANA target promoters in vivo. Moreover, a cell line carrying the methylation mimetic mutant KSHV showed reduced viral gene expression relative to controls both in latency and in the course of reactivation. These results suggest that residue 20 is important for modulation of a subset of LANA functions and properties of this residue, including the hydrophobic character induced by arginine methylation, may contribute to the observed effects.

Involvement of SSRP1 in Latent Replication of Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (also named human herpesvirus 8) is a gamma-herpesvirus that undergoes both lytic and latent infection. During latent infection, two viral elements are required: latency-associated nuclear antigen (LANA), which functions as an origin binding protein, and the latent origin, which resides within the terminal repeats (TRs) of the viral genome. Previously, we identified two cis-elements within the TRs which are required for latent DNA replication: two LANA binding sites (LBS1 and LBS2 [LBS1/2]) and a GC-rich replication element (RE) upstream of LBS1/2. To further characterize the RE, we constructed a 71-bp minimal replicon (MR) and performed a detailed mutational analysis. Our data indicate that the first 8 nucleotides within the RE are critical for replication. Moreover, both the position and the distance between the RE and LBS1/2 can affect origin replication activity, suggesting that the RE may function as a loading pad for cellular proteins involved in replication. Using biotinylated DNA fragments of wild-type or mutant MRs as probes, we identified 30 proteins that preferentially bind to the origin. Among these proteins, structure-specific recognition protein 1 (SSRP1), a subunit of the FACT complex, and telomeric repeat binding factor 2 (TRF2) formed complexes with LANA at the MR region. Furthermore, the small interfering RNA-based knockdown of SSRP1, but not the dominant-negative-based knockdown of TRF2, significantly decreased the efficiency of LANA-dependent DNA replication. These results indicate that SSRP1 is a novel cellular protein involved in LANA-dependent DNA replication.

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.

Analysis of Viral cis Elements Conferring Kaposi's Sarcoma-Associated Herpesvirus Episome Partitioning and Maintenance

Maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) episomes in latently infected cells is dependent on the latency-associated nuclear antigen (LANA). LANA binds to the viral terminal repeats (TR), leading to recruitment of cellular origin recognition complex proteins. Additionally, LANA tethers episomes to chromosomes via interactions with histones H2A and H2B (A. J. Barbera et al., Science 311:856-861, 2006). Despite these molecular details, less is known about how episomes are established after de novo infection. To address this, we measured short-term retention rates of green fluorescent protein-expressing replicons in proliferating lymphoid cells. In the absence of antibiotic selection, LANA significantly reduced the loss rate of TR-containing replicons. Additionally, we found that LANA can support long-term stability of KSHV replicons for more than 2 months under nonselective conditions. Analysis of cis elements within TR that confer episome replication and partitioning revealed that these activities can occur independently, and furthermore, both events contribute to episome stability. We found that replication-deficient plasmids containing LANA binding sites (LBS1/2) exhibited measurable retention rates in the presence of LANA. To confirm these observations, we uncoupled KSHV replication and partitioning by constructing hybrid origins containing the Epstein-Barr virus (EBV) dyad symmetry for plasmid replication and KSHV LBS1/2. We demonstrate that multiple LBS1/2 function in a manner analogous to that of the EBV family of repeats by forming an array of LANA binding sites for partitioning of KSHV genomes. Our data suggest that the efficiency with which KSHV establishes latency is dependent on multiple LANA activities, which stabilize viral genomes early after de novo infection.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Modulates K1 Expression through Its cis -Acting Elements within the Terminal Repeats

K1 is the first open reading frame encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and lies positionally to the immediate right of the terminal repeats. K1 is a transmembrane glycoprotein having a functional immunoreceptor tyrosine-based activation motif (ITAM) capable of activating B-cell receptor signaling. K1 is expressed mostly during the lytic cycle of the virus and its promoter lies within the terminal repeat which contains the binding sites for latency-associated nuclear antigen (LANA). The K1 promoter (K1p) having LANA binding sites assayed by reporter assay demonstrated that LANA is capable of down-regulating K1 promoter transcriptional activity. However, the KSHV replication transcription activator RTA up-regulates K1p transcriptional activity. The promoter deleted of LANA binding sites showed loss in LANA-mediated down-regulation but was unaffected for RTA-mediated up-regulation. Increasing amounts of RTA rescued LANA-mediated repression of K1p transcriptional activity in cotransfection experiments. Reporter assay data suggest that LANA binding to its cognate sequence is critical for LANA-mediated repression of K1p as a LANA construct lacking the DNA binding domain was unable to repress K1p transcription. Additionally, KSHV primary infection experiments suggest that K1 is expressed during early infection but is repressed on the establishment of latency and so follows an expression profile similar to that of RTA during infection. Analysis of the promoter sequence revealed the presence of Oct-1 transcription factor binding sites within the -116 to +76 region. Mutational analysis of the Oct-1 sites abolished RTA-mediated transcriptional activation, suggesting that RTA up-regulates K1p transcription through binding to this transcription factor.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces a Strong Bend on Binding to Terminal Repeat DNA

During latency, the Kaposi's sarcoma-associated herpesvirus genome is maintained as a circular episome, replicating in synchrony with host chromosomes. Replication requires the latency-associated nuclear antigen (LANA) and an origin of latent DNA replication located in the viral terminal repeats, consisting of two LANA binding sites (LBSs) and a GC-rich sequence. Here, we show that the recruitment of a LANA dimer to high-affinity site LBS-1 bends DNA by 57 degrees and towards the major groove. The cooccupancy of LBS-1 and lower-affinity LBS-2 induces a symmetrical bend of 110 degrees . By changing the origin architecture, LANA may help to assemble a specific nucleoprotein structure important for the initiation of DNA replication.

Characterization of the Minimal Replicator of Kaposi's Sarcoma-Associated Herpesvirus Latent Origin

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) binds to two sites within the 801-bp-long terminal repeat (TR) and is the only viral protein required for episomal maintenance. While two or more copies of TR are required for long-term maintenance, a single TR confers LANA-dependent origin activity on plasmid DNA. Deletion mapping revealed a 71-bp-long minimal replicator containing two distinctive sequence elements: LANA binding sites (LBS1/2) and an adjacent 29- to 32-bp-long GC-rich sequence which we termed the replication element. Furthermore, the transcription factor Sp1 can bind to TR outside the minimal replicator and contributes to TR's previously reported enhancer activity.