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. 2013 Jun;87(12):6782-93.
doi: 10.1128/JVI.00011-13. Epub 2013 Apr 10.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen regulates the KSHV epigenome by association with the histone demethylase KDM3A

Kevin Y Kim  1 , Steve B Huerta, Chie Izumiya, Don-Hong Wang, Anthony Martinez, Bogdan Shevchenko, Hsing-Jien Kung, Mel Campbell, Yoshihiro Izumiya
Affiliations

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen regulates the KSHV epigenome by association with the histone demethylase KDM3A

Kevin Y Kim et al. J Virol. 2013 Jun.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) latent genomes are tethered to host histones to form a minichromosome also known as an "episome." Histones, which are core components of chromatin, are heavily modified by various histone-targeting enzymes. Posttranslational modifications of histones significantly influence accessibility of transcriptional factors and thus have profound effects on gene expression. Recent studies showed that epigenetic marks on the KSHV episome are well organized, exemplified by the absence of histone H3 lysine 9 (H3K9) methylation, a heterochromatic histone mark, from immediate early and latent gene promoters in naturally infected cells. The present study revealed a mechanistic insight into KSHV epigenome regulation via a complex consisting of LANA and the H3K9me1/2 histone demethylase JMJD1A/KDM3A. This complex was isolated from HeLa cell nuclear extracts stably expressing LANA and was verified by coimmunoprecipitation analyses and with purified proteins. LANA recruitment sites on the KSHV genome inversely correlated with H3K9me2 histone marks in naturally infected cells, and methylation of H3K9 significantly inhibited LANA binding to the histone H3 tail. Chromatin immunoprecipitation coupled with KSHV tiling arrays identified the recruitment sites of the complex, while depletion of LANA expression or overexpression of a KDM3A binding-deficient mutant decreased KDM3A recruitment to the KSHV genome. Finally, ablation of KDM3A expression from latently KSHV-infected cells significantly inhibited KSHV gene expression, leading to decreased KSHV replication during reactivation. Taken together, our results suggest that LANA may play a role in regulation of epigenetic marks on the KSHV genome, which is in part through association with the histone demethylase KDM3A.

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Figures

Fig 1
Fig 1
KDM3A interacts with KSHV LANA in vivo. (A) Generation of stably 3×Flag-LANA-expressing HeLa cells. Immunoblotting was performed with anti-Flag antibody to confirm LANA expression. Cellular GAPDH was probed with anti-GAPDH monoclonal antibody and used as a loading control. (A) Mass spectrometry analysis. The LANA protein complex was isolated from HeLa nuclear extracts. 3×Flag-tagged LANA was stably expressed in HeLa cells, and nuclear extracts were prepared from 8 liters of suspension culture. Stably Flag-expressing control cells were similarly prepared for a background control. Protein complexes were subjected to SDS-PAGE on a 4-to-16% gradient gel, and the gel was stained with Sypro ruby. Some of the interacting partners that were identified by at least three unique peptides and a total of more than 10 reads with a confidence interval equal to 100% (Mascot/Scaffold program) are listed. hnRNPK, heterogeneous nuclear ribonucleoprotein K. (B) Coimmunoprecipitation of KDM3A and LANA in 293T cells (a) or BCBL-1 cells (b). HEK 293T cells were transiently cotransfected with HA-KDM3A and the Flag control or Flag-LANA. Cell lysates (500 μg) were immunoprecipitated with Flag agarose beads and immunoblotted with anti-HA (a). BCBL-1 cell lysates (1 mg) were immunoprecipitated with rabbit IgG control or anti-KDM3A rabbit IgG and immunoblotted with anti-LANA rat IgG (b). W.B., Western blotting. (C) Colocalization. Immunofluorescence staining of anti-LANA (green) and anti-KDM3A (red) in naturally infected BCBL-1 cells shows colocalization (yellow). Images were taken by using a wide-field 3D deconvolution fluorescence microscope, and 3D image visualization and quantitation were performed by using the VoloCITY digital imaging suite.
Fig 2
Fig 2
In vitro interaction between KDM3A and LANA. (A) Flag-KDM3A and Flag-LANA proteins were purified from baculovirus-infected Sf9 cells. Purified proteins were subjected to SDS-PAGE and stained with Coomassie brilliant blue. (B) Schematic diagram of the domains of LANA. Deletion proteins used in this study are depicted in the middle. GST-LANA proteins were subjected to SDS-PAGE and stained with Coomassie (bottom). An in vitro GST pulldown assay was performed by incubation of purified full-length Flag-KDM3A with immobilized LANA-GST deletion mutants in binding buffer. LANA residues 191 to 251 precipitated nearly 25% of the input of full-length KDM3A. (C) Schematic diagram of the KDM3A domains based on the SMART database. Successive N-terminal GST deletion mutants of KDM3A, prepared from bacterial cells, were incubated with full-length baculovirus-purified Flag-LANA for a series of in vitro GST pulldown assays. The interaction was probed with anti-Flag antibody, and GST proteins used in these studies were visualized by Coomassie staining. (D) Confirmation of the interacting domain in HEK 293T cells. The indicated plasmids were cotransfected into HEK 293T cells. The coprecipitated deletion mutant of KDM3A was probed with anti-GFP antibody. Whole-cell lysates (WCL) were similarly probed and used as input controls.
Fig 3
Fig 3
LANA preferentially binds to the nonmethylated N-terminal H3 tail. Peptide pulldown analyses were performed by incubation of 1 μg of biotinylated peptides with purified LANA, luciferase, or EHMT1. Biotinylated peptides were precipitated with a streptavidin bead mixture and washed extensively with binding buffer containing 500 mM NaCl. Interaction with the histone N-terminal tail was probed by immunoblotting with anti-Flag antibody. The EHMT1 protein was used in the peptide pulldown assay as a positive control for H3K9me1/2 binding, while luciferase protein served as a negative control. LANA but not luciferase interacts with the histone H3 N-terminal tail in a posttranslational-modification-specific manner. The band intensities are shown as a percentage of the input.
Fig 4
Fig 4
LANA and KDM3A recruitment sites on the KSHV genome. (A) LANA and KDM3A occupancy on the KSHV genome was examined by tiling array analyses. Co-occupying regions with more than 5-fold enrichment of both proteins are marked with orange shading. (B) Confirmation of ChIP-on-chip analyses. qPCR was performed on the samples with independent ChIP analyses. LANA and KDM3A were recruited to LANA loci but not the ORF64 coding region, which inversely correlated with the histone H3K9me2 mark. (C) Effects of LANA knockdown on KDM3A recruitment. Efficacy of LANA knockdown was examined by immunoblotting (a), and effects on viral copy number were monitored before ChIP analyses (b). Occupancy of LANA and KDM3A were examined by ChIP analysis followed by qPCR using primers for the LANA promoter and ORF64 coding region (c).
Fig 5
Fig 5
Histone H3K9me2 demethylation of the KSHV genome by KDM3A. (A) KDM3A knockdown from K-Rta-inducible TREx-MH-K-Rta BCBL-1 cell lines. Immunoblot analysis was performed with the indicated antibodies. GAPDH was used as a loading control. (B) ChIP assays. ChIP assays were performed with the indicated antibodies with either shControl or shKDM3A stable TREx-MH-K-Rta BCBL-1 cell lines. Immunoprecipitated DNA was analyzed by qPCR analysis using specific primer pairs for the indicated loci.
Fig 6
Fig 6
Effects of KDM3A knockdown on the KSHV life cycle. (A) Viral gene expression. KSHV reactivation was triggered by incubation with doxycycline for 4 h. Total RNA was harvested at the indicated time points, and KSHV PCR array analysis (a) or individual RT-qPCR (b) was performed to monitor viral gene expression. Cellular GAPDH and actin were used for internal controls, and the mean normalized expression (MNE) level is shown. For array analyses, individual gene expression levels were normalized to the lowest expression level for each column, and fold induction of viral gene expression is shown as a heat map. (B) Viral protein expression. A series of immunoblot analyses was performed to examine the expression of KSHV immediate early, early, and late genes at the protein level. The indicated antibodies were used to probe viral protein expression. Cellular GAPDH was used as a loading control. (C) Viral replication. The intracellular viral DNA copy number was measured by qPCR. The actin coding region was used as an internal control.
Fig 7
Fig 7
Effects of the dominant negative LANA Δ191–251 mutant on the viral life cycle and KDM3A recruitment. (A) Viral gene expression. rKSHV.219-infected HEK 293T cells were transfected with either the empty Flag vector alone or Flag–LANA Δ191–251. At 24 h posttransfection, cells were treated with a combination of TPA (20 nM) and sodium butyrate (3 mM), and lysates were subjected to immunoblotting analyses at the indicated time points. Immunoblotting was performed by using the indicated antibodies. (B) ChIP assays. (a) Immunoblot analysis of rKSHV-infected HEK 239T cells after transient transfection of the indicated plasmids. (b) Transfected cells were used for ChIP analyses using anti-Flag M2 antibody or anti-HA antibody. Wt, wild type.
Fig 8
Fig 8
Hypothetical model of KDM3A and LANA interactions. During latency, the recruitment of KDM3A through LANA interactions maintains a euchromatin state (histones marked in red) on a subset of viral genes by preventing possession of the repressive H3K9me1/2 histone marks that form heterochromatin (histones marked in blue). Depletion of these repressive marks sets the stage for efficient lytic reactivation.
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References

    1. Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM. 1995. Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N. Engl. J. Med. 332:1186–1191 - PubMed
    1. Dupin N, Fisher C, Kellam P, Ariad S, Tulliez M, Franck N, van Marck E, Salmon D, Gorin I, Escande JP, Weiss RA, Alitalo K, Boshoff C. 1999. Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Proc. Natl. Acad. Sci. U. S. A. 96:4546–4551 - PMC - PubMed
    1. Hu J, Garber AC, Renne R. 2002. The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus supports latent DNA replication in dividing cells. J. Virol. 76:11677–11687 - PMC - PubMed
    1. Kelley-Clarke B, De Leon-Vazquez E, Slain K, Barbera AJ, Kaye KM. 2009. Role of Kaposi's sarcoma-associated herpesvirus C-terminal LANA chromosome binding in episome persistence. J. Virol. 83:4326–4337 - PMC - PubMed
    1. Kelley-Clarke B, Ballestas ME, Komatsu T, Kaye KM. 2007. Kaposi's sarcoma herpesvirus C-terminal LANA concentrates at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes. Virology 357:149–157 - PubMed

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