The latency-associated nuclear antigen encoded by Kaposi's sarcoma-associated herpesvirus activates two major essential Epstein-Barr virus latent promoters

Abstract

The latency-associated nuclear antigen (LANA) encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for B-lymphocyte transformation, is expressed. EBNA2 upregulates the expression of LMP1 and other cellular genes through specific interactions with cellular transcription factors tethering EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we demonstrated that LANA can efficiently transactivate both the LMP1 and C promoters in the human B-cell line BJAB as well as in the human embryonic kidney 293 cell line. Moreover, we demonstrated that specific domains of LANA containing the putative leucine zipper and the glutamic acid-rich region are highly effective in upregulating these viral promoters, while the amino-terminal region (435 amino acids) exhibited little or no transactivation activity in our assays. We also specifically tested truncations of the LMP1 promoter element and showed that the -204 to +40 region had increased levels of activation compared with a larger region, -512 to +40, which contains two recombination signal-binding protein J kappa binding sites. The smaller, -204 to +40 promoter region contains specific binding sites for the Ets family transcription factor PU.1, transcription activating factor/cyclic AMP response element, and Sp1, all of which are known to function as activators of transcription. Our data therefore suggest a potential role for LANA in regulation of the major EBV latent promoters in KSHV- and EBV-coinfected cells. Furthermore, LANA may be able to activate transcription of viral and cellular promoters in the absence of EBNA2, potentially through association with transcription factors bound to their cognate sequences within the -204 to +40 region. This regulation of viral gene expression is critical for persistence of these DNA tumor viruses and most likely involved in mediating the oncogenic process in these coinfected cells.

FIG. 1

Schematic diagram of LANA showing its structural properties. Note that the clone consisting of amino acids 1 to 435 contains the proline-rich region (P-rich), a nuclear localization sequence (NLS), and part of the acidic region (AD). The LANA construct consisting of amino acids 301 to 942 contains the entire acidic domain, including the glutamine-rich region (Q-rich) and the entire putative leucine zipper (L-zip). The amino acid 762 to 1162 clone contains the putative leucine zipper and a potential nuclear localization sequence. The LANA construct with amino acids 1 to 756 lacks the putative leucine zipper but contains the acidic domains, the proline-rich region, and a nuclear localization sequence. The amino acid 1 to 950 clone is identical to the amino acid 1 to 756 construct except that it includes the putative leucine zipper.

FIG. 2

LANA transactivation of the major latent EBV promoters, Cp and LMP1, in BJAB cells. The pGL-Cp (A) and pGL-LMP1–512/+72 (B) reporter plasmids (5 μg each) were transfected alone, with 7.5 μg of EBNA2, or with 5, 10, or 15 μg of LANA into 10⁷ BJAB cells as indicated. Equal amounts of cell lysates were assayed for luciferase activity. Luciferase activity is expressed as fold activation relative to that obtained with the pGL promoter alone.

FIG. 3

LANA transactivation of the major latent EBV promoters, Cp and LMP1, in HEK 293 cells. The pGL-Cp (A) and pGL-LMP1–512/+72 (B) reporter plasmids (5 μg each) were transfected alone, with 7.5 μg of EBNA2, or with 5, 10, or 15 μg of LANA into 10⁷ 293 cells as indicated. Equal amounts of cell lysates were assayed for luciferase activity. Luciferase activity is expressed as fold activation relative to that obtained with pGL-Cp alone.

FIG. 4

LANA is expressed when transiently transfected into BJAB and HEK 293 cells. BJAB (A) or HEK 293 (B) cells were transfected with vector alone or with LANA, and slides were prepared and analyzed by immunofluorescence microscopy; a KSHV-infected positive-control cell line for LANA, BC-3, is also shown (C). (D) HEK 293 cells transfected with either vector alone or LANA were electrophoresed on a 6% polyacrylamide–SDS gel and analyzed by Western blotting. LANA-specific signal is seen slightly above the 215-kDa molecular marker. αLANA was obtained from polyclonal rabbit serum made against the carboxy-terminal 300 amino acids of the LANA protein.

FIG. 5

Schematic diagram of the truncated LMP1–512/+40 promoter elements. The −272/+40 construct does not contain the AML1a consensus sites or one Jκ site that is present in the −512/+40 promoter. The −204/+40 promoter is missing these elements as well as an additional Jκ consensus site. All of the promoter truncations contain the PU.1, ATF/CRE, and Sp1 sites.

FIG. 6

The −204/+40 LMP1 promoter element is upregulated by LANA in both BJAB and HEK 293 cells. pGL-LMP1–512/+40, −272/+40, or −204/+40 reporter plasmid (5 μg each) was transfected alone or with 20 μg of LANA into 10⁷ BJAB (A) or HEK 293 (B) cells. Luciferase activity is shown as fold activation relative to that obtained with each of the promoters alone. Equal amounts of cell lysates were assayed for luciferase activity.

FIG. 7

The central glutamine-rich region as well as the carboxy terminus of LANA is involved in transactivation of the LMP1 promoter. (A) pGL-LMP1–512/+40 (5 μg) was transfected alone or with 20 μg of one of the following LANA clones: full-length LANA (FL), LANA amino acids 1 to 435 (1–435), LANA amino acids 301 to 942 (301–942), or LANA amino acids 762 to 1162 (762–1162). (B) pGL-LMP1–512/+40 (5 μg) was transfected alone or with 20 μg of LANA 1–756 or LANA 1–950. Luciferase activity is shown as fold activation above that attained with the promoter alone. Equal amounts of cell lysates were assayed for luciferase activity. (C) pGL-LMP1–204/+40 (5 μg) was transfected alone or with 20 μg of full-length LANA, LANA 1–435, LANA 301–942, or LANA 762–1162. (D) pGL-LMP1–204/+40 (5 μg) was transfected alone or with 20 μg of LANA 1–756 or LANA 1–950. (E) The LANA constructs are expressed when transiently transfected into BJAB cells. Slides were prepared and analyzed by immunofluorescence microscopy, as indicated, 20 h after 20 μg of LANA construct was transfected into BJAB cells. A KSHV-positive control cell line, BC-3, is also shown, as is BJAB transfected with promoter alone as a negative control.

FIG. 8

Expression of LANA, EBNA2, and LMP1 in cells singly infected with EBV and in coinfected PEL cell lines. BJAB is an EBV-negative cell line, B958 and LCL-1 are infected with EBV only, and BC-1 and BC-2 are PEL cell lines coinfected with KSHV and EBV. LANA was fractionated on a 6% polyacrylamide–SDS gel. Polyclonal human serum specific for the LANA protein was used as the primary antibody. EBNA2 and LMP1 were fractionated on an 8% polyacrylamide–SDS gel and detected with EBNA2- and LMP1-specific monoclonal antisera (PE2 and S12, respectively). Protein extracts from BJAB, B958, and LCL-1 cells (5 × 10⁵ each) were fractionated, while 10⁶ BC-1 and BC-2 cells were electrophoresed in lanes 4 and 5, respectively.

FIG. 9

LMP1 is upregulated in P3HR-1 EBV-positive cells transiently transfected with LANA. P3HR-1 cells contain an EBV genome from which EBNA2 has been deleted. BJAB is an EBV-negative cell line used as a negative control. LCL-1 is an EBV-positive, EBNA2-positive cell line. (A) A Western blot for LMP1 prepared using the S12 monoclonal antibody. Lanes 4 to 7 contain lysates from P3HR-1 cells transfected with increasing amounts of LANA. (B) Verification of loading of equivalent levels of protein by comparing band intensities with that of a nonspecific band. (C) A Western blot for LANA prepared using polyclonal human antiserum which is specific for LANA. Lanes 5 to 8 contain lysates from P3HR-1 cells transfected with increasing amounts of LANA and correspond to lanes 4 to 7 in panel A. The positive-control cell line BC-3 exhibits a slightly weaker LANA signal. The transfected LANA construct was cloned from the BC-1 KSHV-positive cell line. P3HR-1 cells without LANA were transfected with vector alone in lane 3 (A and B) or lane 4 (C).