ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) has an etiologic role in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA). LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA's role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease.
Project description: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.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects host cells and establishes lifelong persistence as an extra-chromosomal episome in the nucleus. To persist in proliferating cells, the viral genome typically replicates once per cell cycle and is distributed into daughter cells. This process involves host machinery utilized by KSHV, however the underlying mechanisms are not fully elucidated. In present study, we found that N-Myc downstream regulated gene 1 (NDRG1), a cellular gene known to be non-detectable in primary B cells and endothelial cells which are the major cell types for KSHV infection in vivo, was highly upregulated by KSHV in these cells. We further demonstrated that the high expression of NDRG1 was regulated by latency-associated nuclear antigen (LANA), the major viral latent protein which tethers the viral genome to host chromosome and plays an essential role in viral genome maintenance. Surprisingly, knockdown of NDRG1 in KSHV latently infected cells resulted in a significant decrease of viral genome copy number in these cells. Interestingly, NDRG1 can directly interact with proliferating cell nuclear antigen (PCNA), a cellular protein which functions as a DNA polymerase clamp during DNA replication. Intriguingly, we found that NDRG1 forms a complex with LANA and PCNA and serves as a scaffold protein bridging these two proteins. We further demonstrated that NDRG1 is critical for mediating LANA to recruit PCNA onto terminal repeat (TR) of KSHV genome, and facilitates viral DNA replication and episome persistence. Taken together, our findings suggest that NDRG1 plays an important role in KSHV viral genome replication, and provide new clues for understanding of KSHV persistence.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA's ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.
Project description: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.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) genomes are tethered to the host chromosomes and partitioned faithfully into daughter cells with the host chromosomes. The latency-associated nuclear antigen (LANA) is important for segregation of the newly synthesized viral genomes to the daughter nuclei. Here, we report that the nuclear mitotic apparatus protein (NuMA) and LANA can associate in KSHV-infected cells. In synchronized cells, NuMA and LANA are colocalized in interphase cells and separate during mitosis at the beginning of prophase, reassociating again at the end of telophase and cytokinesis. Silencing of NuMA expression by small interfering RNA and expression of LGN and a dominant-negative of dynactin (P150-CC1), which disrupts the association of NuMA with microtubules, resulted in the loss of KSHV terminal-repeat plasmids containing the major latent origin. Thus, NuMA is required for persistence of the KSHV episomes in daughter cells. This interaction between NuMA and LANA is critical for segregation and maintenance of the KSHV episomes through a temporally controlled mechanism of binding and release during specific phases of mitosis.
Project description:Many pathogens, including Kaposi's sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens.
Project description: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.
Project description:The molecular basis for the formation of functional, higher-ordered macro-molecular domains is not completely known. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) genome forms a super-molecular domain structure during latent infection that is strictly dependent on the DNA binding of the viral nuclear antigen LANA to the viral terminal repeats (TR). LANA is known to form oligomeric structures that have been implicated in viral episome maintenance. In this study, we show that the LANA oligomerization interface is required for the formation of higher-order nuclear bodies that partially colocalize with DAXX, EZH2, H3K27me3, and ORC2 but not with PML. These nuclear bodies assemble at the periphery of condensed cellular chromosomes during mitotic cell division. We demonstrate that the LANA oligomerization interface contributes to the cooperative DNA binding at the viral TR and the recruitment of ORC to the viral episome. Oligomerization mutants failed to auto-regulate LANA/ORF73 transcription, and this correlated with the loss of a chromosome conformational DNA-loop between the TR and LANA promoter. Viral genomes with LANA oligomerization mutants were subject to genome rearrangements including the loss of subgenomic DNA. Our data suggests that LANA oligomerization drives stable binding to the TR and formation of an epigenetically stable chromatin architecture resulting in higher-order LANA nuclear bodies important for viral genome integrity and long-term episome persistence.
Project description:Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.