Kaposi's sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castleman's disease (CD), Kaposi's sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1,704-bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues, including PEL and CD but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV-infected B cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL, or CD as measured by Western blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U2OS cells. While LANA2 specifically interacts with amino acids 290 to 393 of p53 in glutathione S-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.

FIG. 1

Northern hybridization of BC-1 mRNA with an ORFK10.5 probe. Probe hybridization for mRNA from uninduced BC-1 cells (lane 1), BC-1 cells treated with 20 ng of TPA/ml for 48 h (lane 2), BC-1 cells treated with 0.5 mM PFA (lane 3), BC-1 cells treated with 20 ng of TPA/ml and 0.5 mM PFA (lane 4), and KSHV-negative, EBV-infected P3HR1 cells treated with 20 ng of TPA/ml (lane 5) are shown. The probe hybridizes to a 1.8-kb band which is not induced by TPA nor inhibited by PFA treatment, consistent with a latent pattern of viral gene transcription. The same blot is stripped and reprobed with β-actin as a control for loading.

FIG. 2

Transcript map of LANA2 in KSHV genomic environs showing V1 (vIRF1), V2, V3, and V4 probes used for Northern blot hybridization. The V3 probe was used to screen a TPA-induced BC-1 cDNA library. Six phages (φ672, φ701, φ702, φ703, φ731, and φ741) were isolated containing inserts of various sizes. One full-length, 1,735-bp cDNA starting at nt 91425 and terminating at nt 89599 was identified and sequenced from phage φ703. This cDNA contained a start ATG at position 91393 and a splice donor/acceptor site corresponding to nt 90938/90837.

FIG. 3

(A) Comparison of motif domains between IRF4/Pip and LANA2. IRF4 encodes a 450-aa protein with an N-terminal DNA-binding domain (DBD) defined by five tryptophan residues. This characteristic is not found in LANA2 (567 aa); however, a 213-bp region of LANA2, between aa 432 and 503, shows 32% amino acid identity with the C-terminal interaction domain (IAD) of IRF4. (B) Phylogenetic tree for KSHV and human IRF proteins. LANA2 is most closely related to vIRF1 and vIRF2, suggesting a common origin from an ancestral IRF-like gene. Amino acid sequences were aligned using ClustalW, and the phylogenetic tree was generated using the Bootstrap NJ tree 1000 program. Protein peptide sources (GenBank accession numbers) are as follows: hIRF1 [87992], hIRF2 [539621], hIRF3 [4504725], hIRF4 [2497445], hIRF5 [4504727], hIRF6 [3122293], hIRF7 [4809288], ICSBP(hIRF8) [6016308], ISGF3γ [266392], KSHV vIRF1 [4929348], KSHV vIRF2 [3152728], and KSHV LANA2 [AY008303]. A phylogenetic tree comparing the IRF-like proteins from the RRV26-95 isolate and the KSHV IRF-like proteins has been published by Alexander et al (1).

FIG. 4

Cytospin preparation of TPA-stimulated BCBL-1 cells immunostained with CM-10A2 mouse monoclonal antibody against LANA2. LANA2 demonstrates a finely speckled nuclear pattern exclusive of nucleolar zones in essentially all BCBL-1 cells (magnification, ×60 hematoxylin counterstain).

FIG. 5

Immunofluorescence double colocalization of LANA1 and LANA2 in KSHV-infected BCBL-1 cells. (A) LANA1 protein (red) in a coarsely speckled nuclear distribution. (B) Diffuse, finely speckled nuclear pattern of LANA2 protein (green). (C) Double filter; colocalization of LANA1 and LANA2. (A, B, C) Although some subnuclear regions show the distinct dispersal of the two proteins exclusive of each other, yellow nuclear staining is also evident in other areas, possibly representing colocalization of a subfraction of LANA1 and LANA2. Cells undergoing mitosis (arrow) appear to express only LANA1 exclusive of LANA2 (C) (magnification, ×100; Texas red and fluorescein isothiocyanate).

FIG. 6

Immunolocalization of LANA1 compared with LANA2 in KSHV-infected disorders. Shown are LANA1 immunolocalization in a pericardial PEL-infiltrating cardiac muscle (A), a germinal center from a lymph node with multicentric Castleman's disease (B), and a cutaneous KS lesion biopsy (C). Adjacent sections of the same tissues are immunostained for LANA2 (D, E, and F). All tumor cells in PEL express both LANA1 (A) and LANA2 (D), and the majority of the KSHV-infected mantle zone lymphocytes in CD express both LANA1 (B) and LANA2 (E). However, while the majority of KS spindle cells express LANA1 (C), none express LANA2 (F).

FIG. 7

Immunolocalization of LANA1 (A) compared with LANA2 (B) in a lymph node with CD as well as KS. While the KS spindle cells (area within guide lines) and some of the mantle zone lymphocytes show strong nuclear positivity to LANA1, the adjacent section immunostained with LANA2 only shows this protein expressed in the lymphocyte subpopulation of KSHV-infected cells in the mantle. The CD serves as an internal positive control for the negative LANA2 immunostaining of KS spindle cells.

FIG. 8

Inhibition of p53 transcriptional activity by LANA2. A representative luciferase assay shows inhibition of reporter gene expression by transient transfection of pcDNA.LANA2. (A) SAOS-2 cells were transfected with 2 μg of plasmid pG13-Luc reporter plasmid together with 0.0 or 0.5 μg of pcDNA.p53 and 0.5 or 1 μg of pcDNA.LANA2, as indicated. For control, SAOS-2 cells were transfected with the reporter plasmid pGL3-control and 0.0, 0.5, or 1 μg of pcDNA.LANA2. (B) U2OS cells were transfected with 2 μg of plasmid pG13-Luc reporter plasmid with or without 0.5 or 1 μg of pcDNA.LANA2 and were treated with 0.4 μM doxorubicin.

FIG. 9

In vitro GST pull-down assays using [³⁵S]methionine-labeled LANA2 or p53. LANA2 interacts with full-length p53 protein as well as the p53 region between aa 290 and 393.

FIG. 10

LANA2 inhibits p53-induced apoptosis. SAOS-2 cells were transfected with pEGFP-F* and the empty expression vector pCDNA3.1 (A), pCDNA.p53 (B), or pCDNA.p53 and pCDNA.LANA2 (C). Total DNA in all transfections was normalized using the empty expression vector. After 48 h, cells were fixed and stained with propidium iodide. The cellular DNA content was analyzed by flow cytometry. U2OS cells were transfected with pEGFP-F* and the empty expression vector pcDNA (D and E) or pcDNA.LANA2 (F). Eighteen hours after transfection, cells were treated with doxorubicin (0.4 μM) (E and F), and the cells were processed for DNA content analysis 30 h posttreatment. Numbers indicate the percentage of cells in the sub-G¹ phase of the cell cycle.