Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Jul;78(14):7299-310.
doi: 10.1128/JVI.78.14.7299-7310.2004.

Accumulation of heterochromatin components on the terminal repeat sequence of Kaposi's sarcoma-associated herpesvirus mediated by the latency-associated nuclear antigen

Shuhei Sakakibara  1 , Keiji Ueda, Ken Nishimura, Eunju Do, Eriko Ohsaki, Toshiomi Okuno, Koichi Yamanishi
Affiliations

Accumulation of heterochromatin components on the terminal repeat sequence of Kaposi's sarcoma-associated herpesvirus mediated by the latency-associated nuclear antigen

Shuhei Sakakibara et al. J Virol. 2004 Jul.

Abstract

In the latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
LANA colocalizes with HP-1 in BC-3 cells. (A and B) Paraformaldehyde-fixed BC-3 cells were reacted with a mouse monoclonal antibody against HP-1α (red) and serum from an AIDS-KS patient that was used to detect LANA (green). (B) A higher magnification of the image (×400). Arrowheads show representative colocalization of these proteins. (C) LANA does not colocalize with HP-1α in the absence of the KSHV genome. 293 cells in which LANA and DsRed-HP-1α were transiently coexpressed were fixed and reacted with a mouse monoclonal antibody against LANA (generated in our laboratory).
FIG. 2.
FIG. 2.
LANA interacts with SUV39H1. (A) Pull-down assay with recombinant MBP-SUV39H1 and its mutants. The constructs are shown on the right. NE from BC3 cells was incubated with 4 μg of MBP-LacZ (lane 2) and with MBP-fused SUV39H1 (lane 3) and its mutants (39-ΔN [aa 113 to 412] [lane 4], 39-SET [aa 250 to 412] [lane 5], and 39-Chromo [aa 1 to 112] [lane 6]). Each precipitate was probed with an anti-LANA antibody (Advanced Biotechnologies Inc.) and an anti-MBP antibody (New England Biolabs Inc.). Asterisks indicate the MBP fusion proteins. WB, Western blotting. (B) Mapping the site of LANA that was required for the interaction with SUV39H1. Truncated LANA mutants (left panel) were expressed in 293 cells and subjected to the pull-down assay described for panel A (1 μg of recombinant proteins was used for each experiment). Each precipitate was probed with an anti-V5 antibody (Invitrogen) to detect wild-type protein (WT), LNΔN, and D1; with a mouse anti-LANA antibody (generated in our laboratory) to detect LANA; and with rabbit polyclonal anti-GFP antibodies (Medical and Biological Laboratory) to detect GFP-N1 and GFP-NE. The asterisk shows a nonspecific band seen in the MBP-LacZ α precipitation.
FIG. 3.
FIG. 3.
Triangular interaction among LANA, TR DNA, and SUV39H1. (A) Biotinylated-DNA pull-down assay. 293 cells were transfected with expression vectors for LANA (wild type [wt]) or its mutant (LNΔN), which lacked the SUV39H1-interacting site, and pCMV5-flagSUV39H1. Biotinylated DNA of either the LBS or the MCS of pBS-SKII (Stratagene) as a control DNA fragment was incubated with NE from the transfectants and bound to streptavidin-Sepharose (Amersham Bioscience). Precipitates were analyzed on Western blots (WB) probed with a mouse anti-V5 monoclonal antibody and a mouse anti-FLAG monoclonal antibody. The DNA sequence of the LBS is shown. (B) IFA of 293LANA cells with or without the BAC-TR construct. The focal concentration of LANA was detected in 293LANA cells carrying BAC-TR. (C) Colocalization of LANA with SUV39H1 in 293 cells bearing the artificial KSHV DNA (BAC-TR). 293LANA cells were transiently transfected with pCMV5-FLAG-SUV39H1 with (panels 1 to 3) or without (panels 4 to 6) BAC-TR. (D) Association between LANA and heterochromatin in Vero cells stably expressing GFP-LANA with BAC-TR or the control BAC construct (BAC-neor). Parental Vero cell (panels 1 to 3), Vero/gfpLANA cells with the empty BAC (panels 4 to 11), and Vero/gfpLANA cells with BAC-TR (panels 12 to 15) were stained with an anti-HP-1α antibody (panels 1, 4, 8, and 12). The IFA was performed as described in Materials and Methods.
FIG. 4.
FIG. 4.
Repression of gene expression by LANA in a reporter plasmid harboring the TR. (A) Schematic representation of the reporter plasmids used in the transient-transfection assay and a representative structure of the KSHV genome and its genes (top). Part of the TR-containing fragment from cosmid Z6 (EcoRI-BglII) was linked to the RTA-promoter luciferase cassette. Z6, cloning region in cosmid Z6 (NIH AIDS Research and Reference Reagent Program); Amp R, ampicillin resistance gene; Luc, luciferase gene. (B) Transient assay for transcriptional regulation by LANA of a TR-bearing plasmid. The transfection and luciferase assay were performed as described in Materials and Methods. 293 cells were transfected with the effector plasmid (total of 2 μg/well in a 12-well plate) and the reporter plasmid (0.2 μg/well). (C) SUV39H1 enhances the repression by LANA. 293 cells were cotransfected transiently with pTriEX-LANA with pFLAG-SUV39H1 (0.3 and 0.6 μg/well) or pFLAG-SUV39H1ΔN (SUV39ΔN) (0.3 and 0.6 μg/well). The luciferase activities were normalized to the protein concentration and are shown in the upper graph, in which the open bars indicate empty vector (pTriEX1.1) (0.5 μg/well) and the closed bars indicate pTriEX-LANA (0.5 μg/well). Transfection efficiency could not be normalized to a reference plasmid such as a β-galactosidase expression vector due to LANA's modulation of some promoters. The fold repression by LANA was calculated as follows and is shown in the lower graph: [(pTriEXLANA + X)/(pTriEX + X)]−1, where the designations in parentheses show the luciferase units (relative light units [RLU]) for each combination and X is pFLAGCMV1, pFLAG-SUV39H1, or pFLAG-SUV39H1ΔN (SUV39ΔN). The assays shown in panels B and C were performed in triplicate for each set of transfections, and the mean value and standard deviation were calculated. (D) Western blotting (WB) of 293 cells transfected with SUV39H1 and its mutant expression vector. The expression of each construct (0.6 μg/well) transfected with pFLAGCMV1 (lane 1), pFLAG-SUV39H1 (lane 2), and pFLAG-SUV39ΔN (lane 3) in 293 cells was analyzed by Western blotting with an anti-FLAG antibody (Sigma Aldrich).
FIG. 5.
FIG. 5.
ChIP. (A) ChIP and Western blotting (WB) with BC-3 cells. In vivo cross-linked chromatin was digested with micrococcal nuclease (DNA length of < 1kb) and immunoprecipitated with 1 μg of a rat anti-LANA antibody or control rat immunoglobulin (Dako). The precipitated matrix was analyzed by Western blotting with an anti-LANA antibody, an anti-HP-1α antibody (Euromedex), and an anti-HP-1β antibody (Euromedex). IgL, light chain of immunoglobulin. (B) Chromatin states of the TR and the K1 and LANA promoters. PCR was performed with primers for the TR, the K1 gene, and the promoter of the latent transcript (LANA) after ChIP. The amplified products were separated by electrophoresis in a 2% agarose gel and stained with ethidium bromide (Inp, 5% of input; C, control rat immunoglobulin; LN, rat anti-LANA monoclonal antibody [Advanced Biotechnologies Inc.]; HP, mouse anti-HP-1α monoclonal antibody [Upstate]; H3MeK9, mouse anti-methylated histone H3 monoclonal antibody [Upstate]). (C) The KSHV ORF50/RTA region was specifically precipitated by the anti-HP-1α antibody in the ChIP assay. Precipitated DNA was reacted in the PCR with the primers for the ORF50/RTA locus (panels 1 and 2) or the ORF73/LANA locus (panels 3 and 4), and the results of 2% agarose-TBE electrophoresis are shown. A schematic presentation of KSHV ORF50/RTA and ORF73/LANA is also shown. Arrows indicated the priming site of each PCR primer.
FIG. 6.
FIG. 6.
Working hypothesis for viral transcriptional control by LANA in latency. (a) First, LANA binds with the TR DNA sequence in the KSHV genome and recruits the SUV39H1 histone methyltransferase. SUV39H1 methylates lysine K9 in the amino terminus of histone H3, where HP-1 is specifically recruited. (b) HP-1 then interacts with SUV39H1, resulting in the methylation of neighboring histones. (c) Finally, the heterochromatin architecture spreads over the viral genome. Some genes might be suppressed if their regulatory elements lie in the heterochromatin region.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aagaard, L., G. Laible, P. Selenko, M. Schmid, R. Dorn, G. Schotta, S. Kuhfittig, A. Wolf, A. Lebersorger, P. B. Singh, G. Reuter, and T. Jenuwein. 1999. Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J. 18:1923-1938. - PMC - PubMed
    1. Ballestas, M. E., P. A. Chatis, and K. M. Kaye. 1999. Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284:641-644. - PubMed
    1. Ballestas, M. E., and K. M. Kaye. 2001. Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mediates episome persistence through cis-acting terminal repeat (TR) sequence and specifically binds TR DNA. J. Virol. 75:3250-3258. - PMC - PubMed
    1. Bannister, A. J., P. Zegerman, J. F. Partridge, E. A. Miska, J. O. Thomas, R. C. Allshire, and T. Kouzarides. 2001. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410:120-124. - PubMed
    1. Bell, A. C., A. G. West, and G. Felsenfeld. 1999. The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98:387-396. - PubMed

Publication types

MeSH terms

Associated data