Human herpesvirus 8 LANA interacts with proteins of the mSin3 corepressor complex and negatively regulates Epstein-Barr virus gene expression in dually infected PEL cells

Abstract

The human herpesvirus 8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in all latently HHV-8 infected cells and in HHV-8-associated tumors, including primary effusion lymphoma (PEL). To better understand the contribution of LANA to tumorigenesis and to the PEL phenotype, we performed a yeast two-hybrid screen which identified the corepressor protein SAP30 as a LANA binding protein. SAP30 is a constituent of a large multicomponent complex that brings histone deacetylases to the promoter. Glutathione S-transferase affinity assays confirmed interaction between LANA and SAP30 and also demonstrated interactions between LANA and two other members of the corepressor complex, mSin3A and CIR. The corepressors bound to the amino-terminal 340-amino-acid domain of LANA. In transient expression assays, this same domain of LANA mediated repression when targeted to a 5xGal4tk-CAT reporter as a GAL4-LANA fusion. PEL cells have the unusual feature that they are frequently dually infected with both HHV-8 and Epstein-Barr virus (EBV). We found that EBV EBNA-1 expression is downregulated in PEL cells at both the RNA and protein levels. In transient expression assays, LANA repressed activated expression from the EBV Qp and Cp latency promoters. Reduction of endogenous Qp activity could also be demonstrated in EBV-infected Rael cells transfected with a LANA expression plasmid. In contrast to the effect of LANA on EBV latency promoters, LANA activated expression from its own promoter. The data indicate that LANA can mediate transcriptional repression through recruitment of an mSin3 corepressor complex and further that LANA-mediated repression is likely to contribute to the low level of EBV latency gene expression seen in dually infected PEL cells.

FIG. 1

LANA binds to SAP30. Shown are results of a yeast two-hybrid assay in which interaction is measured by induction of beta-galactosidase activity. Yeast cells were cotransformed with Gal4DBD-LANA and Gal4ACT-SAP30, Gal4DBD-LANA plus Gal4ACT vector (negative control), or Gal4DBD-CIR plus Gal4ACT-SAP30 (positive control). Data are averages from two experiments, with the range of values indicated.

FIG. 2

LANA interacts with the corepressors SAP30, mSin3A, and CIR in GST affinity assays. (A) Diagram of the GST-LANA constructs used in the affinity assay. (B) Extracts from HeLa cells transfected with Myc-SAP30 were incubated with the indicated GST-LANA fusion proteins (lanes 1 to 4) or with control GST protein (lanes 5 and 6). Transfected cell extract (15 μl) was loaded in lane 7. A longer exposure (lanes 6 and 7) was needed to detect Myc-SAP30 in the cell extract. (C) Extracts from HeLa cells transfected with Myc-mSin3A or CIR-Flag were incubated with the indicated GST-LANA fusion proteins (lanes 1 to 3) or with control GST protein, control GST-Zta, or GST-EBNA2 protein (lanes 4 to 6). Transfected cell extract (15 μl) was loaded in lane 7. Bound protein was separated by SDS-PAGE and subjected to Western blot analysis with either an anti-Myc or an anti-Flag antibody. Reactive proteins were visualized by chemiluminescence.

FIG. 3

SAP30 colocalizes with LANA in transfected cells. An indirect immunofluorescence assay was performed on Vero cells transfected with Flag-LANA (A), Myc-SAP30 (B), or Myc-SAP30 plus Flag-LANA (C and D). In singly transfected cells, Flag-LANA (A) exhibited micropunctate staining with nucleolar sparing and Myc-SAP30 (B) exhibited nuclear punctate plus diffuse nucleolar staining. The doubly transfected cells were stained for Myc-SAP30 (C) (green) or Flag-LANA (D) (red). In cotransfected cells, Myc-SAP30 (C) exhibited the same micropunctate distribution as Flag-LANA (D). Myc-SAP30 (green) was detected using a rabbit anti-Myc primary antibody and FITC-conjugated donkey anti-rabbit immunoglobulin secondary antibody. Flag-LANA (red) was detected using a mouse monoclonal anti-Flag primary antibody and a rhodamine-conjugated donkey anti-mouse secondary antibody.

FIG. 4

LANA acts as a repressor of gene expression. (A) Diagram of the Gal4-LANA fusion constructs used in the reporter assay. (B) Transient expression assay in which HeLa cells were transfected with the tk-luciferase control plasmid (1.0 μg) and a 5×Gal4BS-tkCAT reporter (1.5 μg) alone or in the presence of the indicated Gal4-LANA fusion plasmids (1 μg). Fusion proteins containing LANA aa 1 to 340 repressed reporter CAT expression as efficiently as full-length LANA(1-1177). The assay was repeated three times with similar results.

FIG. 5

EBV EBNA-1 protein expression is reduced in dually infected PEL cells. Shown are Western blot analyses comparing the expression of EBNA-1 in EBV-positive cell lines and HHV-8 and EBV dually infected cell lines. B95-8, Raji, and Akata are EBV⁺ B-cell lines. HBL-6 and BC-2 are HHV-8⁺ EBV⁺ PEL cell lines. BCBL-1 is an HHV-8⁺ EBV⁻ PEL cell line, and DG75 is negative for both viruses. EBNA-1 was detected by using the anti-EBNA-1 monoclonal antibody EBNA.OT1x (A) or NPC serum (B). Reactive proteins were visualized by using chemiluminescence.

FIG. 6

EBNA-1 mRNA expression is also reduced in PEL cells. A semiquantitative RT-PCR assay for EBNA-1 transcripts was performed using the indicated serial 10-fold dilutions of the template cDNA. RT-PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and photographed. B95-8 and Raji are EBV⁺ B-cell lines; HBL-6 and BC-2 are EBV⁺ HHV-8⁺ PEL cell lines. Control RT-PCR results for cellular GAPDH mRNA are shown for the Raji and HBL-6 cell lines.

FIG. 7

LANA attenuates Qp-driven expression of EBNA-1. (A) Transient expression assay in which HeLa cells were transfected with a Qp-CAT reporter (1 μg) alone or with JAK-1 (2 μg) and in the presence or absence of cotransfected LANA (1 μg). The results shown are averages from three experiments, with the standard deviations indicated. (B) LANA reduces Qp-driven EBNA-1 protein expression in Rael cells. Shown is a Western blot examining EBNA-1 expression in EBV-negative DG75 cells carrying the pEBO vector, which expresses EBNA-1 (lane 1), EBV-positive Rael cells carrying the pEBO vector and grown for 11 weeks in selection medium containing hygromycin B (lane 2), Rael cells (lane 3), Rael pEBO-LANA cells grown for 3 weeks in selection medium (lane 4), Rael pEBO-LANA cells after 7 weeks of selection (lane 5), and Rael pEBO-LANA cells after 11 weeks of selection (lane 6). The pEBO-encoded EBNA-1 differs in mobility from the endogenous Rael EBNA-1. EBNA-1 was detected using NPC serum and a chemiluminescence visualization protocol. (C) LANA expression in transfected Rael cells. Shown is a Western blot in which LANA was detected using a rat anti-LANA monoclonal antibody (ABI). Lane 1, Rael pEBO cells after 11 weeks of selection; lane 2, Rael cells; lane 3, Rael pEBO-LANA cells after 11 weeks of selection. Rael pEBO-LANA cells express LANA, unlike the parental Rael or control Rael pEBO cells.

FIG. 8

In transient assays LANA represses EBV Cp-driven expression but activates the HHV-8 LANAp. CAT and luciferase reporter assays were performed in transfected HeLa cells. Results shown are averages from three experiments, with the standard deviations indicated. (A) Cells were transfected with the Cp-CAT reporter (1.0 μg), EBNA-2 (0.3 μg), LANA (2.0 μg), or EBNA-2 (0.3 μg) plus increasing amounts of LANA (0.5, 1.0, or 2.0 μg) as indicated. LANA did not affect basal Cp activity but abolished EBNA-2-induced expression. (B) Cells were transfected with the LANAp-luciferase reporter (1.0 μg), alone or together with LANA (2.0 μg) or LANA(1-275) (2.0 μg) as indicated. LANA positively regulated its own promoter.