Induction of Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen by the lytic transactivator RTA: a novel mechanism for establishment of latency

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jkappa (RBP-Jkappa), as a RBP-Jkappa-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jkappa. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.

FIG. 1.

Scheme showing the LANA promoter and RTA protein. (A) Scheme showing the LANA promoter used in the present study. Putative transcriptional factor binding sites are shown, and numbers indicate nucleotides according to the KSHV genome from BC-1 (26, 29-31, 54). (B) As shown, RTA is a 691-amino-acid protein (BC-1 strain). Numbers indicate amino acids. The putative domains include an N-terminal DNA binding domain and C-terminal activation domain, proline rich domain (P-rich), and leucine zipper (L-zip); RTA also has two NLSs in both the amino- and carboxy-terminal regions. The approximate position of each functional interacting domain is shown by a black bar, corresponding to the interacting partner shown in the column on the right (23, 24, 39-42, 55, 62).

FIG. 2.

Immnoblotting to check KSHV virion-associated protein. (A) Cell lysate of BJAB (lane 1), uninduced BCBL1 (lane 2), TPA induced BCBL1 (lane 3), trypsin and Triton X-100-treated virions (lane 4), trypsin-treated virions (lane 5), and mock-treated virions (lane 6) were separated by an 8% SDS-PAGE gel, transferred to an nitrocellulose membrane, and then blotted with RTA mouse monoclonal antibody, anti-LANA rabbit serum, ORF45 mouse monoclonal antibody, and anti-gB rabbit serum. (B) Cell lysate of BJAB (lane 1), uninduced BCBL1 (lane 2), TPA-induced BCBL1 (lane 3), supernatant of trypsin-treated purified virions (lane 4), supernatant of Triton X-100-treated virions (lane 5), and virion pellet-treated by trypsin plus Triton X-100 (lane 6) were separated on an 8% SDS-PAGE gel, transferred to an nitrocellulose membrane, and then blotted with RTA mouse monoclonal antibody, anti-LANA rabbit serum, ORF45 mouse monoclonal antibody and anti-gB rabbit serum.

FIG. 3.

Transcriptional activity of RTA on LANA promoters in cells. The reporter plasmid pGLLANAp contains an 800-bp sequence upstream of the start code of the LANA gene that drives the expression of firefly luciferase (29-31). A fixed amount (5 μg) of the reporter plasmids was transfected or cotransfected into 15 million U2OS (A), BJAB (B), and 293 (C) cells with 2.5 μg, 5 μg, 10 μg, 15 μg, and 20 μg of pCR3.1-RTA. At 24 h posttransfection, cell lysate of each transfection was harvested for the luciferase assay. The promoter activity was expressed as the fold activation relative to the reporter-alone control. The means and standard deviations from three independent transfections are shown.

FIG. 4.

A fixed amount of reporter plasmid pGLLANAp was transfected into 15 million RTA-inducible BCBL1 cells (A) and control cells (B), at different time points 0 h, 12 h, 24 h, 36 h, 48 h, and 72 h postinduction with tetracycline (5 μg/ml), and cell lysate was harvested for a luciferase assay. The promoter activity was expressed as the fold activation relative to the reporter-alone control. The means and standard deviations from three independent transfections are shown.

FIG. 5.

RTA induces LANA expression. (A) A total of 15 million T-antigen-transformed human embryonic kidney 293 cells were transfected with 20 μg L54 DNA and increasing amounts of RTA expression vector pCR3.1-RTA (lane 1 to lane 6); the amount of pCR3.1-RTA was 0 μg, 2.5 μg, 5 μg, 10 μg, 15 μg, or 20 μg. The total transfected DNA was normalized with pCR3.1 vector. At 24 h posttransfection, protein lysates were analyzed by Western blotting for levels of expression of transfected protein with LANA rabbit serum to detect LANA and RTA monoclonal antibody for detection of RTA protein. (B) RTA was induced in 50 million RTA-inducible BCBL1 cells with 5 μg of tetracycline/ml. Total RNA was collected from the cells at 0, 12, 24, 48, and 72 h postinduction. A total of 5 μg of RNA was used with the Superscript First Strand Synthesis system to construct cDNA. Real-time PCR was performed using the DyNAmo SYBR Green qPCR kit with β-actin as the standard. The PCR data are expressed as the C_t values at each time point for RTA and LANA. Each time point was tested in triplicate for the calculation of the mean and standard deviation. (C) The relative transcript abundance based on the C_t values for RTA and LANA.

FIG. 6.

Real-time RT-PCR analysis of RTA and LANA transcripts in 293 cells infected with KSHV. Total RNA was isolated from 20 million 293 cells infected with virus from 50 million BCBL1 cells at 30 min and 1, 3, 5, 8, 12, 16, 20, 24, and 48 h postinfection by TRIzol reagent. A total of 5 μg of RNA was used with the Superscript First Strand Synthesis system to construct cDNA. Real-time PCR was performed using the DyNAmo SYBR Green qPCR kit with β-actin as the standard. (A) cDNA samples for RTA. RTA transcript abundance as calculated by C_t values; the standard curve is shown to the right. (B) cDNA samples for LANA. LANA transcript abundance as calculated by the C_t values, with standard curve to the right. Each time point and standard were tested in triplicate for the calculation of the mean and standard deviation.

FIG. 7.

Comparison of activity of WT and ΜΤ (RBP-Jκ binding site mutated) LANA promoter. A fixed amount (5 μg) of the WT or MT reporter plasmid was transfected or cotransfected into 15 million BJAB (A) and U2OS (B) cells with 2.5 μg, 5 μg, 10 μg, 15 μg, or 20 μg of pCR3.1-RTA. At 24 h posttransfection, the cell lysate of each transfection was harvested for a luciferase assay. (C) WT LANA promoter reporter plasmid (5 μg) was transfected into a RBP-Jκ knockout cell line (OT11) and a wild-type cell line (OT13) with 10 μg pCR3.1-RTA expression vector; an extra rescue experiment was carried out introducing the addition of RBP-Jκ expression vector into OT11. At 24 h posttransfection, cell lysate of each transfection was harvested for a luciferase assay.

FIG. 8.

RTA/RBP-Jκ DNA binding complex. The probe for the EMSA consists of a RBP-Jκ consensus binding sequence and the flanking bases from the LANA promoter. An arrow indicates the position of the RBP-Jκ/DNA complex. Asterisks indicate the position of the supershifted complex in the presence of RTA- and RBP-Jκ-cotransfected 293 cell lysate or in vitro-translated mixture of RTA and RBP-Jκ with specific antibodies. Non, nonspecific; IVT, in vitro translated; NE, nuclear extract.

FIG. 9.

LANA expression is delayed in OT11 cells. OT13 (A) and OT11 (B) cells were infected by KSHV for 2 h and then washed once with PBS. Cells were incubated and harvested at 4 h, 12 h, 24 h, and 48 h and fixed with 1:1 methanol/acetone. Latent protein LANA and lytic protein RTA were detected by immunofluorescence assay.

FIG. 10.

A hypothetical model for regulation of LANA by RTA contributing to latency establishment postinfection.