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
. 2002 Nov;76(22):11596-604.
doi: 10.1128/jvi.76.22.11596-11604.2002.

Protein interactions targeting the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus to cell chromosomes

Anita Krithivas  1 , Masahiro Fujimuro, Magdalena Weidner, David B Young, S Diane Hayward
Affiliations

Protein interactions targeting the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus to cell chromosomes

Anita Krithivas et al. J Virol. 2002 Nov.

Abstract

Maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) latent infection depends on the viral episomes in the nucleus being distributed to daughter cells following cell division. The latency-associated nuclear antigen (LANA) is constitutively expressed in all KSHV-infected cells. LANA binds sequences in the terminal repeat regions of the KSHV genome and tethers the viral episomes to chromosomes. To better understand the mechanism of chromosomal tethering, we performed glutathione S-transferase (GST) affinity and yeast two-hybrid assays to identify LANA-interacting proteins with known chromosomal association. Two of the interactors were the methyl CpG binding protein MeCP2 and the 43-kDa protein DEK. The interactions of MeCP2 and DEK with LANA were confirmed by coimmunoprecipitation. The MeCP2-interacting domain was mapped to the previously described chromatin binding site in the N terminus of LANA, while the DEK-interacting domain mapped to LANA amino acids 986 to 1043 in the C terminus. LANA was unable to associate with mouse chromosomes in chromosome spreads of transfected NIH 3T3 cells. However, LANA was capable of targeting to mouse chromosomes in the presence of human MeCP2 or DEK. The data indicate that LANA is tethered to chromosomes through two independent chromatin binding domains that interact with different protein partners.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
LANA localization on chromosomes of KSHV-infected cells. Chromosome spreads of BCBL1 cells in which the LANA punctate spots (green) localize separately from centromeres (red doublets). Inset, higher magnification of the merged image.
FIG. 2.
FIG. 2.
MeCP2 targets LANA to sites of heterochromatin. NIH 3T3 cells were transfected with GFP-LANA and either Flag-HP1α or Flag-MeCP2. Immunofluorescence assays revealed that (A) GFP-LANA (green) did not localize to sites of mouse heterochromatin marked by Flag-HP1α staining (red). (B) GFP-LANA (green) relocalized to heterochromatin in the presence of Flag-MeCP2 (red). (C) GFP-LANA (green) localized to the Flag-MeCP2 marked pericentromeric regions of mouse NIH 3T3 chromosomes (red spots) in the presence of MeCP2. Inset, higher magnification of the merged image. (D) Chromosome spreads of NIH 3T3 cells transfected with GFP-LANA (green) reveal no association between LANA and mouse chromosomes. DNA was stained with DAPI (blue).
FIG. 3.
FIG. 3.
MeCP2 targets LANA to chromosomes via the N-terminal CBS. (A) Schematic of wild-type (wt) LANA N terminus showing the CBS and NLS. The CBS is disrupted in mtLANA. (B) Immunoprecipitation assay with extracts of HeLa cells cotransfected with Flag-MeCP2 and either mtLANA or wild-type LANA. mtLANA did not coprecipitate with Flag-MeCP2 (lane 1), unlike wild-type LANA (lane 3). Neither mtLANA nor wild-type LANA was precipitated by control mouse IgG (lanes 2 and lane 4). Lanes 5 to 8 show transfected cell extracts (15 μl; lanes 5 and 6) and direct precipitation by anti-LANA antibody (lanes 7 and 8). ms Ab, mouse antibody. (C to E) Immunofluorescence assays performed with NIH 3T3 cells transfected with GFP-LANA-N or GFP-mtLANA and Flag-MeCP2. (C) LANA-N (green) localized to sites of heterochromatin in the presence of MeCP2 (red). (D) mtLANA (green) did not colocalize with heterochromatin in the presence of MeCP2 (red). (E) Chromosome spreads revealed that Flag-MeCP2 (red) localized to chromosomes, while mtLANA (green) failed to associate with chromosomes. DNA was stained with DAPI (blue).
FIG. 4.
FIG. 4.
LANA C-terminus associates independently with chromosomes. Immunofluorescence assay showing chromosome spreads of HeLa cells transfected with GFP-LANA-C (green spots), which associated with human chromosomes. DNA was stained with DAPI (blue). Inset, higher magnification of the merged image.
FIG. 5.
FIG. 5.
LANA binds to DEK through a C-terminal domain. (A) Yeast two-hybrid assay in which interaction is measured by induction of β-galactosidase activity. Yeast cells were cotransformed with Gal4DBD-LANA plus Gal4ACT-DEK, Gal4ACT vector (negative control), or Gal4ACT-SAP30 (positive control). Data are averaged from two experiments, with the percent error indicated. (B) In vitro-translated [35S]methionine-labeled DEK was incubated with the indicated GST-LANA fusion proteins (lanes 1 to 3) or with control GST-EBNA2(1-58) (GST-E2) or GST proteins (lanes 4 and 5). Reaction extract (2 μl) was loaded in lane 6. Bound protein was separated by SDS-PAGE and analyzed by autoradiography for 4 h. (C) Western blot of immunoprecipitates from extracts of HeLa cells cotransfected with Flag-LANA or Flag-LANA-C and Myc-DEK and probed with anti-Flag antibody. Flag-LANA (lane 1) and Flag-LANA-C (lane 3) coprecipitated with Myc-DEK, but were not precipitated by control mouse IgG (lanes 2 and 4). Lanes 5 and 6, transfected cell extracts (15 μl). Extract in the input lanes was one-fifth of that used for coprecipitations. (D) The upper panel shows a schematic of the Flag-tagged LANA deletion mutants used in the GST affinity assay. The lower panel shows in vitro-translated, [35S]methionine-labeled LANA mutants m1 to m4 were incubated with a GST-DEK fusion protein (lanes 1, 4, 7, and 10) or with control GST protein (lanes 2, 5, 8, and 11). Extract (2 μl) was loaded in lanes 3, 6, 9, and 12. Bound protein was separated by SDS-PAGE and analyzed by autoradiography for 24 h.
FIG. 6.
FIG. 6.
DEK interacts with mouse chromosomes. (A) NIH 3T3 cells were transfected with GFP-DEK and Flag-MeCP2. GFP-DEK (green) did not localize to sites of heterochromatin, unlike Flag-MeCP2 (red). (B) Chromosome spreads of NIH 3T3 cells transfected with GFP-DEK and Flag-MeCP2. GFP-DEK (green) painted mouse chromosomes and did not localize to the regions of heterochromatin marked by MeCP2 (red). Inset, higher magnification of the merged image.
FIG. 7.
FIG. 7.
DEK can independently mediate LANA chromosomal tethering. Chromosome spreads of NIH 3T3 cells (A) and HeLa cells (B) transfected with GFP-mtLANA and Myc-DEK. Myc-DEK (red) targeted GFP-mtLANA (green) lacking the N-terminal CBS to mouse and human chromosomes. (C) Chromosome spreads of wild-type (wt) LANA versus mtLANA in HeLa cells. mtLANA lacking the N-terminal CBS does not associate with chromosomes. DNA was stained with DAPI (blue).
FIG. 8.
FIG. 8.
Model of LANA-mediated chromosomal tethering. LANA is targeted to chromosomes through N-terminal interactions with MeCP2, which binds methylated CpG dinucleotides in intranucleosomal linker DNA, and through C-terminal interactions with DEK, which associates with core histone proteins. Chromosomal association mediates episomal tethering through binding of the LANA C terminus to KSHV DNA (5, 10, 22).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexiadis, V., T. Waldmann, J. Andersen, M. Mann, R. Knippers, and C. Gruss. 2000. The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner. Genes Dev. 14:1308-1312. - PMC - PubMed
    1. An, J., A. K. Lichtenstein, G. Brent, and M. B. Rettig. 2002. The Kaposi sarcoma-associated herpesvirus (KSHV) induces cellular interleukin 6 expression: role of the KSHV latency-associated nuclear antigen and the AP1 response element. Blood 99:649-654. - PubMed
    1. Antequera, F., and A. Bird. 1993. CpG islands, p. 169-185. In J. Jost and H. Saluz (ed.), DNA methylation: molecular biology and biological significance. Birkhauser Verlag, Basel, Switzerland.
    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

Publication types

MeSH terms

LinkOut - more resources