Reactivation and Lytic Replication of Kaposi’s Sarcoma-Associated Herpesvirus: An Update (original) (raw)
Related papers
Molecular Biology of KSHV Lytic Reactivation
Viruses, 2015
Kaposi's sarcoma-associated herpesvirus (KSHV) primarily persists as a latent episome in infected cells. During latent infection, only a limited number of viral genes are expressed that help to maintain the viral episome and prevent lytic reactivation. The latent KSHV genome persists as a highly ordered chromatin structure with bivalent chromatin marks at the promoter-regulatory region of the major immediate-early gene promoter. Various stimuli can induce chromatin modifications to an active euchromatic epigenetic mark, leading to the expression of genes required for the transition from the latent to the lytic phase of KSHV life cycle. Enhanced replication and transcription activator (RTA) gene expression triggers a cascade of events, resulting in the modulation of various cellular pathways to support viral DNA synthesis. RTA also binds to the origin of lytic DNA replication to recruit viral, as well as cellular, proteins for the initiation of the lytic DNA replication of KSHV. ...
KSHV lytic proteins K-RTA and K8 bind to cellular and viral chromatin to modulate gene expression
PLOS ONE
The oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.
KSHV Rta Promoter Specification and Viral Reactivation
Frontiers in Microbiology, 2012
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.
Long Non-Coding RNA and Epigenetic Gene Regulation of KSHV
Viruses, 2014
Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is a γ-herpesvirus linked to Kaposi's sarcoma (KS) and two lymphoproliferative disorders, primary effusion lymphoma (PEL or body-cavity B-lymphoma [BCBL]) and a subset of Multicentric Castleman's Disease. During lytic growth, pervasive viral transcription generating a variety of transcripts with uncertain protein-coding potential has been described on a genome-wide scale in β-and γ-herpesviruses. One class of such RNAs is called long non-coding RNAs (lncRNAs). KSHV encodes a viral lncRNA known as polyadenylated nuclear RNA (PAN RNA), a copious early gene product. PAN RNA has been implicated in KSHV gene expression, replication, and immune modulation. PAN RNA expression is required for optimal expression of the entire KSHV lytic gene expression program. Latent KSHV episomes are coated with viral latency-associated nuclear antigen (LANA). LANA rapidly dissociates from episomes during reactivation. Here we review recent studies suggesting that PAN RNA may function as a viral lncRNA, including a role in the facilitation of LANA-episomal dissociation during lytic replication.
Journal of Virology, 2014
Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and LBS2) on viral DNA and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here, we have examined LANA interactions with host chromatin on a genome-wide scale using chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LANA-binding site 1 (LBS1) motif in KSHV DNA. Comparison of the ChIP-seq profile with whole-transcriptome (high-throughput sequencing of RNA transcripts [RNA-seq]) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. IMPORTANCE Here, we employ complementary genome-wide analyses to evaluate the distribution of the highly abundant latency-associated nuclear antigen, LANA, on the host genome and its impact on host gene expression during KSHV latent infection. Combined, ChIP-seq and RNA-seq reveal that LANA accumulates at active gene promoters that harbor specific short DNA sequences that are highly reminiscent of its cognate binding sites in the virus genome. Unexpectedly, we found that such association does not lead to remodeling of global host transcription during latency. We also report for the first time that LANA's ability to bind host and viral chromatin is highly dynamic and is disrupted in cells undergoing an extensive lytic reactivation. This therefore suggests that the association of LANA to chromatin during a productive infection cycle is controlled by a new regulatory mechanism.
KSHV Genome Replication and Maintenance
Frontiers in Microbiology, 2016
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.
Chromatinization of the KSHV Genome During the KSHV Life Cycle
Cancers, 2015
Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome ...
Journal of Virology, 2008
Lytic reactivation from latency is critical for the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta's direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.
Scientific Reports, 2016
The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization postreplication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANAmediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANAregulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA. Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV8), is linked to Kaposi's sarcoma, primary effusion lymphomas (PELs), and multicentric Castleman's disease, which causes tumors in AIDS patients 1,2. Like other herpesviruses, KSHV displays two distinct life cycles, the default latent and the productive lytic phase, and persists predominantly in the latent form. During latency, only a limited number of viral proteins are expressed, including the latency-associated nuclear antigen encoded by open reading frame 73 (ORF73) 3,4. LANA is expressed in all KSHV-positive tissues and cell lines 5. The full repertoire of viral gene expression occurs during the lytic replication (reactivation) phase, which is likely essential to maintaining the population of newly infected cells and the induction of viral pathogenesis 6. LANA is a nuclear protein with 1162 amino acids and is 220-230 kDa in size. It interacts with various cellular and viral proteins to regulate transcription, cellular signaling, viral DNA replication, and genome maintenance 3. During latent infection, KSHV DNA persists as multi-copy, chromatinized closed circular episomes tethered to the host chromosomes 3,7. LANA binds to the viral genome in the TR region through its carboxyl terminus and attaches to the nucleosomes through the amino terminus for efficient persistence 8,9. LANA, a multifunctional protein, also plays a role in maintaining viral latency, efficient segregation of episomal DNA, and oncogenesis 3,10. LANA has also been shown to modulate the transcription of a variety of cellular and viral promoters 11,12. LANA can serve both as an activator and repressor of gene transcription, suppressing p53 and VHL-driven transcriptions and activating the transcriptions of E2F1 and cyclin-dependent kinase-2 (CDK2) 3,13,14. LANA has been implicated in tumorigenesis through its interactions and interference with cellular pathways associated with cell cycle control, apoptosis, gene expression and immune regulation 15,16. Furthermore, LANA negatively regulates the transcription of viral lytic genes during the establishment of latency 17. It also represses the transcription of RTA, an immediate early gene encoded by ORF50, which activates the switch from
The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes
PLoS Pathogens, 2010
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 tumorderived 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.