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. 2003 Jun;77(12):7093-100.
doi: 10.1128/jvi.77.12.7093-7100.2003.

A Domain in the C-terminal region of latency-associated nuclear antigen 1 of Kaposi's sarcoma-associated Herpesvirus affects transcriptional activation and binding to nuclear heterochromatin

Abel Viejo-Borbolla  1 , Emrah Kati, Julie A Sheldon, Kavita Nathan, Karin Mattsson, Laszlo Szekely, Thomas F Schulz
Affiliations

A Domain in the C-terminal region of latency-associated nuclear antigen 1 of Kaposi's sarcoma-associated Herpesvirus affects transcriptional activation and binding to nuclear heterochromatin

Abel Viejo-Borbolla et al. J Virol. 2003 Jun.

Abstract

The latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the maintenance and replication of viral episomal DNA. The binding sites for nuclear heterochromatin and transcriptional repressor complexes are located in an amino-terminal region of LANA-1, whereas those for viral episomal DNA, p53, pRB, and members of the BRD/fsh family of nuclear proteins are located in its carboxy-terminal domain. LANA-1 activates or represses several cellular and viral promoters. In this report we show that a domain of 15 amino acids (amino acids 1129 to 1143), located close to the carboxy-terminal end of LANA-1, is required for the interaction of LANA-1 with nuclear heterochromatin or nuclear matrix, and for the ability of LANA-1 to activate the Epstein-Barr virus Cp promoter. LANA-1 proteins that are tightly associated with nuclear heterochromatin or matrix differ in molecular weight from LANA-1 proteins that can be dissociated from the nuclear matrix by high-salt buffers, suggesting that posttranslational modifications may determine the association of LANA-1 with nuclear heterochromatin or matrix.

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Figures

FIG. 1.
FIG. 1.
The region from aa 1129 to 1143 of LANA-1 is required for transcriptional activation of the EBV Cp promoter. (A) Binding sites for different nuclear proteins on LANA-1 and other functionally important regions, as far as they have been mapped at present. TR, terminal repeat in the KSHV genome; pRB, retinoblastoma protein; ✽, NLS, nuclear localization signal. (B) Deletion constructs of LANA-1 used in the present study showing the C-terminal 5 aa of each construct. (C) Activation of the EBV Cp promoter by LANA-1 deletion mutants. A total of 50 ng of a reporter construct containing 2.0 kb of EBV sequence upstream of the transcriptional start site of the EBV Cp promoter and a luciferase reporter gene were transfected into 293 cells, together with increasing concentrations of LANA-1, and the luciferase activity was measured. The relative activation of the LANA-1 constructs is shown. The experiment was repeated more than four times. A representative experiment is shown. Expression of the different LANA-1 constructs was checked with KS-positive human sera (not shown). LANA-1* represents a wild-type LANA-1 construct lacking a His tag.
FIG. 2.
FIG. 2.
Intranuclear distribution of LANA-1 deletion mutants. MCF7 cells grown on glass coverslips were transfected with LANA-1 and individual LANA-1 deletion mutants (see Fig. 1 for a description of mutants). At 2 days after transfection, cells were stained with serum from a patient with KS and fluorescence-conjugated secondary antibody. (A) Staining for LANA-1 (green), and DNA (Hoechst 33258), and overlay. (B) Black-and-white photograph showing the difference in nuclear staining pattern between L29 (heterochromatin-associated pattern) and L28 (diffuse nuclear staining).
FIG. 3.
FIG. 3.
Association of LANA-1 and LANA-1 deletion mutants with fraction of the nuclear matrix that is resistant to extraction in high-salt buffers. 293T cells were transfected with LANA-1-expressing constructs, lysed in low-ionic-strength buffer containing 1% NP-40, followed by the addition of a buffer containing 500 mM KCl and the hsr fraction of the nuclear material pelleted by centrifugation as described in the text. After the pellet was washed in high-salt buffer, the hsr pellet and hse supernatant were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, with serum from a Kaposi's sarcoma patient to detect LANA-1. Arrowheads indicate the position of the two different LANA-1 isoforms in the hse and hsr fractions.
FIG. 4.
FIG. 4.
Association of aa 1 to 30 and aa 1128 to 1162 of LANA-1 with nuclear heterochromatin. (A) Diagram of EGFP fusion constructs used in this experiment. The region from aa 1 to 30 of LANA-1, containing a previously described (32) binding site (aa 1 to 24) for nuclear heterochromatin and a nuclear localization signal (aa 24 to 30), was fused upstream of EGFP, by using the vector EGFPN2, and aa 1128 to 1162 of LANA-1, representing its C-terminal end (see Fig. 1) were fused downstream of EGFP by using the vector EGFPC1. MCS, multiple cloning site; NLS, nuclear localization signal. (B) Intranuclear localization of EGFP fusion proteins in L cells. Cells were transfected with the constructs indicated, and the intracellular localization of the corresponding proteins was analyzed by fluorescence. (C) Association of aa 1 to 30 and aa 1128 to 1162 of LANA-1 with the hsr and hse nuclear fractions. The experiment was carried out as described in the legend to Fig. 3, except that an antibody to EGFP was used to stain the Western blots. Arrows indicate the position of EGFP expressed from the pEGFP-N2 (left panel) and pEGFP-C1 (right panel) vectors. Arrowheads indicate the positions of aa1-30/EGFP and EGFP/aa1128-1162 fusion proteins.
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