MLL1 is regulated by KSHV LANA and is important for virus latency.
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ABSTRACT:
SUBMITTER: Tan M
PROVIDER: S-EPMC8682764 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) stabilizes hypoxia-inducible factor α (HIF-1α) during latent infection, and HIF-1α reactivates lytic replication under hypoxic stress. However, the mechanism utilized by KSHV to block lytic reactivation with the accumulation of HIF-1α in latency remains unclear. Here, we report that LANA encoded by KSHV contains a unique SUMO-interacting motif (LANA(SIM)) which is specific for interaction with SUMO-2 and facilitates LANA SUMOylation at lysine 1140. Proteomic and co-immunoprecipitation analysis further reveal that the SUMO-2 modified transcription repressor KAP1 is a critical factor recruited by LANA(SIM). Deletion of LANA(SIM) led to functional loss of both LANA-mediated viral episome maintenance and lytic gene silencing. Moreover, hypoxia reduced KAP1 SUMOylation and resulted in dissociation of both KAP1 and Sin3A repressors from LANA(SIM)-associated complex. Therefore, the LANA(SIM) motif plays an essential role in KSHV latency and is a potential drug target against KSHV-associated cancers.
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Project description:Host signal-transduction pathways are intimately involved in the switch between latency and productive infection of herpes viruses. As with other herpes viruses, infection by Kaposi's sarcoma herpesvirus (KSHV) displays these two phases. During latency only few viral genes are expressed, while in the productive infection the virus is reactivated with initiation of extensive viral DNA replication and gene expression, resulting in production of new viral particles. Viral reactivation is crucial for KSHV pathogenesis and contributes to the progression of KS. We have recently identified Pim-1 as a kinase reactivating KSHV upon over-expression. Here we show that another Pim family kinase, Pim-3, also induces viral reactivation. We demonstrate that expression of both Pim-1 and Pim-3 is induced in response to physiological and chemical reactivation in naturally KSHV-infected cells, and we show that they are required for KSHV reactivation under these conditions. Furthermore, our data indicate that Pim-1 and Pim-3 contribute to viral reactivation by phosphorylating the KSHV latency-associated nuclear antigen (LANA) on serine residues 205 and 206. This counteracts the LANA-mediated repression of the KSHV lytic gene transcription. The identification of Pim family kinases as novel cellular regulators of the gammaherpesvirus life cycle facilitates a deeper understanding of virus-host interactions during reactivation and may represent potential novel targets for therapeutic intervention.
| S-EPMC2648312 | biostudies-literature
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.
| S-EPMC6364946 | biostudies-literature
Project description:Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA's acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.
| S-EPMC7486799 | biostudies-literature
Project description:The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.
| S-EPMC3475679 | biostudies-literature
Project description:Kaposi Sarcoma Herpesvirus (KSHV), a ?2-herpesvirus and class 1 carcinogen, is responsible for at least three human malignancies: Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease (MCD). Its major nuclear latency protein, LANA, is indispensable for the maintenance and replication of latent viral DNA in infected cells. Although LANA is mainly a nuclear protein, cytoplasmic isoforms of LANA exist and can act as antagonists of the cytoplasmic DNA sensor, cGAS. Here, we show that cytosolic LANA also recruits members of the MRN (Mre11-Rad50-NBS1) repair complex in the cytosol and thereby inhibits their recently reported role in the sensing of cytoplasmic DNA and activation of the NF-?B pathway. Inhibition of NF-?B activation by cytoplasmic LANA is accompanied by increased lytic replication in KSHV-infected cells, suggesting that MRN-dependent NF-?B activation contributes to KSHV latency. Cytoplasmic LANA may therefore support the activation of KSHV lytic replication in part by counteracting the activation of NF-?B in response to cytoplasmic DNA. This would complement the recently described role of cytoplasmic LANA in blocking an interferon response triggered by cGAS and thereby promoting lytic reactivation. Our findings highlight a second point at which cytoplasmic LANA interferes with the innate immune response, as well as the importance of the recently discovered role of cytoplasmic MRN complex members as innate sensors of cytoplasmic DNA for the control of KSHV replication.
| S-EPMC5415203 | biostudies-literature
Project description:Shugoshin-1 (Sgo1) protects the integrity of the centromeres, and H2A phosphorylation is critical for this process. The mitotic checkpoint kinase Bub1, phosphorylates H2A and ensures fidelity of chromosome segregation and chromosome number. Oncogenic KSHV induces genetic alterations through chromosomal instability (CIN), and its essential antigen LANA regulates Bub1. We show that LANA inhibits Bub1 phosphorylation of H2A and Cdc20, important for chromosome segregation and mitotic signaling. Inhibition of H2A phosphorylation at residue T120 by LANA resulted in dislocation of Sgo1, and cohesin from the centromeres. Arrest of Cdc20 phosphorylation also rescued degradation of Securin and Cyclin B1 at mitotic exit, and interaction of H2A, and Cdc20 with Bub1 was inhibited by LANA. The N-terminal nuclear localization sequence domain of LANA was essential for LANA and Bub1 interaction, reversed LANA inhibited phosphorylation of H2A and Cdc20, and attenuated LANA-induced aneuploidy and cell proliferation. This molecular mechanism whereby KSHV-induced CIN, demonstrated that the NNLS of LANA is a promising target for development of anti-viral therapies targeting KSHV associated cancers.
| S-EPMC6136811 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates DNA replication of terminal repeat (TR) DNA to enable viral episome persistence in latently infected cells. Southern blotting is routinely used to detect LANA-replicated DNA. We developed and validated a real-time PCR assay for TR-associated DNA and compared it with Southern blot analysis. Both PCR and Southern blot detected LANA-replicated DNA, but the PCR assay was more rapid and did not require radioisotope. PCR detection at 24 and 72 hours post-transfection demonstrated rapid loss of transfected TR DNA. LANA, and to a lesser extent a moderately deficient LANA mutant, reduced the rate of DNA loss through addition of replicated TR DNA and reduction in the loss of non-replicated DNA, the latter of which is consistent with LANA's nuclear segregation function. Therefore, this work develops a rapid, sensitive, and quantitative PCR (qPCR) assay to detect KSHV LANA-replicated DNA and demonstrates that LANA reduces TR DNA loss after transfection through replication and nuclear partitioning of TR DNA.
| S-EPMC3145832 | biostudies-literature
Project description:The humoral antibody response against Kaposi sarcoma-associated herpesvirus (KSHV) in infected individuals has been characterized demonstrating the latency-associated nuclear antigen (LANA) as the most antigenic KSHV protein. Despite the antigenicity of the protein, specific LANA epitopes have not been systematically characterized. Here, we utilized a bacteriophage T7 library, which displays 56-amino acid KSHV LANA peptides with 28-amino acid overlap (VirScan), to define those epitopes in LANA targeted by antibodies from a cohort of 62 sub-Saharan African Kaposi sarcoma (KS) patients and 22 KSHV-infected asymptomatic controls. Intra- and inter-patient breadth and magnitude of the anti-LANA responses were quantified at the peptide and amino acid levels. From these data, we derived a detailed epitope annotation of the entire LANA protein, with a high-resolution focus on the N- and C-termini. Overall, the central repeat region was highly antigenic, but the responses to this region could not be confidently mapped due to its high variability. The highly conserved N-terminus was targeted with low breadth and magnitude. In a minority of individuals, antibodies specific to the nuclear localization sequence and a portion of the proline-rich regions of the N-terminus were evident. In contrast, the first half of the conserved C-terminal domain was consistently targeted with high magnitude. Unfortunately, this region was not included in LANA partial C-terminal crystal structures, however, it was predicted to adopt predominantly random-coil structure. Coupled with functional and secondary structure domain predictions, VirScan revealed fine resolution epitope mapping of the N- and C-terminal domains of LANA that is consistent with previous antigenicity studies and may prove useful to correlate KSHV humoral immunity with pathogenesis.
| S-EPMC9810164 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three malignancies- Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Central to the pathogenesis of these diseases is the KSHV viral life cycle, which is composed of a quiescent latent phase and a replicative lytic phase. While the establishment of latency enables persistent KSHV infection and evasion of the host immune system, lytic replication is essential for the dissemination of the virus between hosts and within the host itself. The transition between these phases, known as lytic reactivation, is controlled by a complex set of environmental, host, and viral factors. The effects of these various factors converge on the regulation of two KSHV proteins whose functions facilitate each phase of the viral life cycle-latency-associated nuclear antigen (LANA) and the master switch of KSHV reactivation, replication and transcription activator (RTA). This review presents the current understanding of how the transition between the phases of the KSHV life cycle is regulated, how the various phases contribute to KSHV pathogenesis, and how the viral life cycle can be exploited as a therapeutic target.
| S-EPMC7551196 | biostudies-literature