Epstein-Barr Virus Inhibits Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication in Primary Effusion Lymphomas^{▿ ¶}

Cell culture, plasmids, adenoviruses, and antibodies.

All cell lines used in this study and their properties are listed in Table 1. Expression plasmids for K-RTA (pCMV50) and the recombinant adenoviruses for green fluorescence protein (GFP) (AdGFP) and K-RTA (AdRTA) were a gift from Byrd Quinlivan (71). The K-RTA K152E mutant is a DNA-binding mutant of K-RTA (91). The recombinant adenovirus for LMP-1 (AdLMP1) was a gift from Stephen Gottschalk and Cliona Rooney (37). The β-galactosidase expression plasmid, pCMVβ, was purchased from Clontech. LMP-1 and LMP-DM, which is a mutant LMP-1 failing to initiate signals, have been described previously (94). The LMP-1 promoter reporter constructs were generated by inserting PCR products into the pGL3-basic vector (Promega). LMP-mISRE-luc is exactly the same as LMP-ISRE-luc except for the interferon-stimulated response element (ISRE) mutation. The LMP-1 ISRE (5′-AGGAAATGGAAAGG-3′) was mutated at the underlined nucleotides for mISRE (5′-AGGGGGTGGGGGGG-3′). The pHP vector was constructed by ligation of the PacI/blunt-XbaI fragment of pAdTrack-HP (96) into the AseI/blunt-XbaI sites of pEGFP-N1 (ClonTech). The sequence 5′-TGGAACGCGACCTTGAGAG-3′ was used to clone the small interfering RNA for LMP-1 (siLMP-1) expression plasmid in pHP. The 5′-TGGCGCAAGATGACAAGGG-3′ sequence was used for making siRNA for K-RTA. The sequence for p53 has been described previously (96). All constructs were verified by sequence analysis by the UNL Sequencing Facility. The K-RTA antibody was from David Lukac (all figures except Fig. 1). Rabbit polyclonal antibody against K-RTA peptide (amino acids 667 to 691), “ATPANEVQESGTLYQLHQWRNYFRD,” was customer made by Alpha Diagnostic International. This K-RTA antibody was used for Fig. 1. The K8 antibody was from Jae Jung. Tubulin antibody was purchased from Sigma. EBV E-RTA and EA-D antibodies were purchased from Argene, LMP-1 antibody (CS1-4) was from Dako, and STAT-1 monoclonal antibody was from Santa Cruz. Rat monoclonal antibody to KSHV latency-associated nuclear antigen was obtained from ABI. Secondary goat antirat conjugated with cy5 was from Jackson ImmunoResearch.

Enrichment of transfected cells and making cell lines.

Enrichment for CD4-positive cells was performed with the use of anti-CD4 antibody conjugated to magnetic beads according to the manufacturer's recommendation (Dynal). The isolated cells were lysed immediately and used for Western blot analysis. However, for chemical treatment, cells were detached from Dynabeads CD4 by incubation for 45 to 60 min at room temperature with 10 μl of DETACHaBEAD (Dynal). The detached beads were removed by using a magnet separation device. The released cells were washed two to three times with 500 μl RPMI 1640 plus 10% fetal bovine serum (FBS) and resuspended in RPMI 1640 plus 10% FBS at 5 × 10⁵ cells/ml. Cells were recovered for 2 to 6 h before the addition of chemical reagents. BC1 (EBV⁺ KSHV⁺) cells were transfected with either pHP (control) or pHP-siLMP-1 (siLMP-1) along with a CD4 expression plasmid, and the transfected cells were enriched and cultured in the presence of G418 (1 mg/ml) and 20% FBS for 1 week to make cell lines. The cells were then treated by chemicals for induction of lytic replication.

Preparation of KSHV stocks and in vitro infections.

BCBL-1 cells were treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) (30 ng/ml) and sodium butyrate (3 mM) to induce the lytic cycle of KSHV replication, and culture supernatants were harvested 7 days later. Virions were pelleted by centrifugation at 100,000 × g for 1 h. Virion pellets were subsequently resuspended in complete medium (1:100 of the volume of the original supernatants) and were passed through a 0.45-μm filter. For infection, 293-EBV or BZLF1-KO cells at approximately 50% confluence were incubated with concentrated viruses. After incubation at 37°C for 24 h, cells were washed and cell lysates were used for detection of target proteins.

Preparation of EBV stocks and in vitro infections.

EBV lytic replication of AGS-BX1-g cells was induced by 0.5 mM sodium butyrate and 30 ng/ml of TPA. Media were replaced with fresh media without TPA and butyrate after 24 h. Media were collected 4 days later with or without concentration and passed through a 0.45-μm filter. For infection, Cro6 cells were incubated with 0.5 or 1 ml of EBV stock. After incubation at 37°C for 24 h, cells were collected and RNA was isolated for reverse transcription-PCR (RT-PCR) analysis. Infectivity was examined by detecting GFP-positive cells under a UV microscope. Similar results were obtained with concentrated or nonconcentrated EBV viruses (data not shown).

Transient transfection, adenovirus infection, and Reporter assays.

Effectene (QIAGEN) was used for the transfection of 293-EBV, BRLF1-KO, and 293T cells. Electroporation was used for transfection of BC3 and DG75 cells. The recombinant adenoviruses were titered in 293 cells. BRLF1-KO cells were infected with adenovirus at a multiplicity of infection of 5 (calculated from PFU), while a multiplicity of infection of 100 was used for infection of BC3 cells. The luciferase assays were performed using the assay kit from Promega according to the manufacturer's recommendation.

EMSA.

Expression and purification of KSHV RTA proteins have been described previously (91). The probes were obtained by first annealing complementary oligonucleotides and then labeling them with [α-³²P]dCTP (Amersham) using DNA polymerase Klenow fragment (Fermentas). The LMP-1 ISRE probe was obtained by annealing two oligonucleotides, 5′-GATCGCAACAGGAAATGGAAAGGC-3′ and its complementary strand, with GATC at the 5′ end. An electrophoretic mobility shift assay (EMSA) was performed essentially as described previously (73, 91).

Induction of KSHV lytic replication.

BC3 cells were treated with 5 to 10 ng/ml TPA for 18 to 24 h to induce lytic viral replication. For BC1 cells, 0.5 to 1 mM sodium butyrate was used for 18 to 24 h for the induction of KSHV lytic replication. For experiments shown in Fig. 1, BC3, BC3-EBV (clone [cl] 10), Cro6, and Cro6-EBV (cl 2) cells were treated with sodium butyrate at 0.5 to 1 mM.

Western blot analysis, RNA extraction, RPA, and RT-PCR.

Standard Western blot analysis was performed as described previously (92). Total RNA was isolated from cells using the RNeasy Total RNA isolation kit (QIAGEN). RNase protection assays (RPA) were performed with total RNA using the RNase Protection Assay Kit II (Ambion) as described previously (93). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was from U.S. Biochemicals, Inc. The probe for LMP-1 was a gift from Joseph Pagano. Reverse transcription was performed with the use of the SuperScript reverse transcription system from Invitrogen, following the manufacturer's recommended protocol. Reverse-transcribed cDNA (2 μl) was then PCR amplified with the appropriate primers. PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 30 to 35 cycles of 95°C for 30 s, 62°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 10 min. Amplified DNA was then resolved by 8% polyacrylamide gel electrophoresis (PAGE). Primers used for LMP-1 were 5′-TGATCATCTTTATCTTCAGAAGAGACCTT-3′ and 5′-CCTGTCCGTGCAAATTCCA-3′; for K-RTA, 5′-CGCAATGCGTTACGTTGTTG-3′ and 5′-GCCCGGACTGTTGAATCG-3′; and for actin, 5′-TTCTACAATGAGCTGCGTGT-3′ and 5′-GCCAGACAGCACTGTGTTGG-3′.

Cell lysates and immunoprecipitation.

Cells (10⁸) were collected by centrifugation and were resuspended in 1 ml of extraction buffer (0.5% NP-40, 0.3 M NaCl, 0.1 mM EDTA, 20 mM HEPES [7.4], 1 mM dithiothreitol, and 10% glycerol), supplemented with complete protease inhibitors (Roche). The cell suspension was kept in 4°C for 30 min with gentle agitation. Insoluble material was pelleted at 14,000 × g for 15 min at 4°C, and the supernatant was used directly for immunoprecipitation. A specific antibody was added to the lysates, followed by addition of 40 μl of protein G Sepharose (Pharmacia), and the sample was incubated overnight at 4°C with continuous mixing. Protein immunocomplexes were isolated by centrifugation, and beads were washed three times with extraction buffer. Immunoprecipitated proteins were resuspended in sodium dodecyl sulfate (SDS)-PAGE loading buffer and analyzed by SDS-PAGE, and Western blot analyses were employed with specific antibodies.

EBV inhibits lytic replication of KSHV.

Because the majority of the current studies on KSHV and EBV use singly infected cells as a model system, the potential effects of EBV and KSHV on each other's biology obviously need attention. We thus examined the effect of EBV on the KSHV lytic replication process. BC3 and Cro6 are KSHV⁺ EBV− PEL lines. Isogenic dually infected cell lines were generated by EBV infection (76). The expression of the entire panel of EBV latent genes, including those for EBV nuclear antigen 1 (EBNA-1), -2, -3, -4, and -6, LMP1, -2A, and -2B, and EBV-encoded small RNAs, was examined with these isogenic cell lines. All EBV-infected clones of the PEL cells expressed EBNA-1, EBV-encoded small RNA 1, and LMP2A. EBNA2 to -6 were not expressed in any of the converted lines. The expression of LMP-1 was heterogeneous as determined by Western blot analyses: Cro6-EBV (cl 2) expressed LMP-1 protein. BC3-EBV (cl 10) had no detectable LMP-1 protein (76). We now have shown that LMP-1 is expressed in both BC3-EBV and Cro6-EBV lines in an RT-PCR assay (see Fig. S1 in the supplemental material). All these data suggested that EBV formed type II latency with low levels of LMP-1 expression in these EBV convertants used in present study, which is in agreement with previous reports about EBV status in dually infected cells (11, 26, 42, 50).

Most isogenic cell lines are made and cultured for some time and have not encountered any major loss of EBV, primarily due to the fact that these EBV-infected PELs are maintained in G418 selection. Because recombinant EBV containing GFP was used for the establishment of isogenic lines, expression of GFP could be used to identify these EBV-positive cells. We found that these EBV convertants were 100% positive for KSHV as determined by the expression of KSHV latency-associated nuclear antigen (also called ORF73) (see Fig. S2A in the supplemental material) and 65% and 85% GFP positive for BC3-EBV and Cro6-EBV, respectively (see Fig. S2B in the supplemental material). Thus, BC3-EBV and Cro6-EBV had 65 and 85% cells, respectively, that were dually infected by both KSHV and EBV.

KSHV lytic replication can be induced by some chemicals, such as sodium butyrate. The cells were treated with the same amounts of sodium butyrate, and KSHV lytic gene expression was examined by Western blot analyses. As shown in Fig. 1, expression of the K-RTA and K8 genes was detected after the treatment, indicating the initiation of lytic replication processes. However, in the presence of EBV, K-RTA and K8 were expressed at lower levels (Fig. 1). Because both K-RTA and K8 are essential genes required for KSHV lytic replication (6, 43, 88), these results suggested that EBV inhibited sodium butyrate-induced KSHV lytic replication.

KSHV RTA induces expression of EBV LMP-1 protein.

The EBV LMP-1 promoter region has three kinds of cis elements through which K-RTA could possibly regulate: RBP-Jκ binding sequence (RBP-Jκ), ISRE, and gamma interferon activation sequence (GAS) (40, 51, 52, 91) (see Fig. 4A). We thus tested if K-RTA could induce the expression of LMP-1. The recombinant adenoviruses containing K-RTA (AdRTA) or GFP (AdGFP) were used to infect a BRLF-1-KO cell line (EBV⁺ KSHV⁻) (27). The EBV BRLF1 gene product (or E-RTA) has been shown to induce the expression of LMP-1 (17). The use of the BRLF1-KO cells would eliminate the potential contribution of EBV BRLF1 to LMP-1 induction. As shown in Fig. 2A, endogenous LMP-1 expression was increased significantly in response to AdRTA infection but not to AdGFP infection.

BRLF1-KO cells are generated in human 293 cells in which recombinant adenovirus can replicate. In order to eliminate the possible contribution from adenovirus replication, we examined the effect of K-RTA in transient-transfection assays. An expression plasmid of K-RTA or the EBV homologue of K-RTA, E-RTA (EBV BRLF1 gene product), was transfected into BRLF1-KO cells. As shown in Fig. 2B, K-RTA could induce the expression of LMP-1; however, E-RTA could not induce the expression of LMP-1 to similar levels. The inability of E-RTA to induce LMP-1 might be due to the differences in cellular background (17). Because LMP-1 is expressed during the EBV lytic replication cycle (8, 20, 67), it is thus possible that K-RTA might induce EBV lytic replication that results in the synthesis of LMP-1. However, this scenario is unlikely because it is well documented that K-RTA is unable to induce the lytic replication of EBV, and we confirmed the observation (74; also data not shown). The results thus suggested that K-RTA-mediated LMP-1 induction is not related to activation of EBV lytic replication.

Induction of the LMP-1 protein by K-RTA is independent of EBNA-2.

Because EBNA-2 is the primary activator of EBV LMP-1 (1, 35, 77, 84), it is possible that K-RTA induces the expression of EBNA-2, which in turn activates LMP-1. P3HR1 (EBV⁺ KSHV⁻) is a Burkitt's lymphoma line that harbors an EBV genome without EBNA-2 (2). K-RTA expression plasmids were transfected into P3HR-1 cells along with a CD4 expression plasmid, and the levels of LMP-1 were determined after selection of the transfected cells by the use of CD4 antibody-conjugated magnetic beads (see Materials and Methods for details). As shown in Fig. 2C, K-RTA induced a marked increase in LMP-1 protein levels in P3HR1 cells. Because of the deletion of EBNA-2 in the P3HR1 EBV genome, these results indicated that the induction of LMP-1 by K-RTA is, at least partially, independent of EBNA-2. The mutant, K-RTA-K152E, was unable to induce LMP-1. K-RTA-K152E also failed to induce the LMP-1 protein in BRLF1-KO cells (data not shown). Because K-RTA-K152E has a defect in DNA binding capability, DNA binding activity of K-RTA might be an important component of the induction of LMP-1 (91). K-RTA also induced the expression of LMP-1 in EBV latency cells with both EBNA2 and BRLF1 in the viral genome, suggesting the induction was not a cell-specific phenomenon.

K-RTA induces EBV LMP-1 at the RNA level.

Next, we examined whether K-RTA induces LMP-1 at the RNA level. K-RTA or its mutant K-RTA-K152E was transfected into BRLF1-KO cells. Total RNA was isolated from transfected cells 24 h later. RPA were used for detection of LMP-1 RNA. K-RTA-transfected cells have higher LMP-1 RNA levels than vector- or K-RTA-K152E-transfected cells (Fig. 3A and B). The expression levels of K-RTA and its mutant were similar (Fig. 3C). Thus, K-RTA-mediated induction of LMP1 occurs at the RNA level.

K-RTA activates LMP-1 promoter.

There are at least three potential cis elements that may be used by RTA for transactivation: RBP-Jκ, ISRE, and GAS (40, 51, 52, 91). K-RTA and various LMP-1 promoter reporter constructs (Fig. 4A) were transfected into 293T cells, and the promoter activities were measured. The expression levels of K-RTA and its mutant were similar (see Fig. S3A in the supplemental material). As shown in Fig. 4B, K-RTA was able to activate empty pGL3basic two- to sixfold under our conditions (columns 1 and 2), which is a background issue. The LMP-ISRE-luc reporter was strongly activated by K-RTA (columns 3 and 4), but the point mutations or deletion of ISRE crippled activation by K-RTA (columns 6 to 9). In agreement with the data shown in Fig. 2C and 3A, the K-RTA-K152E mutant failed to activate LMP-1 promoter reporter constructs (Fig. 4, column 5). Because LMP-ISRE-luc contains ISRE, RBP-Jκ, and GAS (Fig. 4A), these data suggest that the LMP-1 ISRE is a necessary cis element in response to K-RTA in the LMP-1 promoter.

Whether the LMP-1 promoter constructs are responsive to physiological levels of K-RTA was examined. In BC3 (KSHV⁺ EBV⁻) cells, sodium butyrate induced 10-fold activation of LMP-ISRE-luc; however, only two- to threefold activations were observed when pGL3-basic or LMP-mISRE-luc was used for transfection (Fig. 4C). Moreover, in DG75 (EBV⁻ KSHV⁻) cells, sodium butyrate could not activate the LMP-ISRE-luc reporter construct (data not shown). These data suggest that a viral gene(s) in the lytic replication cycle may be responsible for activation of the LMP-1 promoter via LMP-1 ISRE. K-RTA is among the earliest gene products expressed in lytic replication, and its expression was confirmed by Western blot analyses (see Fig. S3B in the supplemental material). Thus, the physiological levels of K-RTA might also activate the LMP-1 promoter construct.

K-RTA binds to LMP-1 promoter.

Finally, because K-RTA is known to bind to the ISRE (91), it is expected that K-RTA could bind to the LMP-1 ISRE. We first tested if the LMP-1 ISRE was able bind to endogenous K-RTA in lysates from PEL cells with lytic replication. However, we did not detect any specific binding (data not shown). Partially purified K-RTA protein, which has been used widely to study K-RTA binding (22, 55, 72, 73, 85), was then used for EMSA with the LMP-1 ISRE probe. The purification process was done according to the method of our previous report, and partially purified K-RTA has the expected properties (91). The same amounts of partially purified proteins were used for EMSA. The binding of K-RTA but not K-RTA K152E to the LMP1 ISRE probe was observed (Fig. 4D). Some bands regularly appeared with our purified K-RTAs. Purified bovine serum albumin was used to detect these nonspecific binding patterns. In addition, cold LMP-ISRE was able to compete for binding to K-RTA, and the K-RTA-DNA complex could be supershifted by the use of K-RTA-specific antibody (see Fig. S3C in the supplemental material). The purified proteins have similar purities as determined by Coomassie blue staining (see Fig. S3D in the supplemental material). These data suggested that K-RTA was able to bind to LMP-1 ISRE specifically in vitro as expected.

LMP-1 negatively regulates the lytic replication of KSHV.

EBV LMP-1 has been shown to suppress EBV reactivation triggered by either anti-immunoglobulin M or TPA (3, 63). In addition, we have shown that LMP-1 is an antiviral protein (87, 90). These published results suggest that LMP-1 might inhibit the lytic replication of KSHV.

BC-3 cells (KSHV⁺ EBV⁻) were transfected with LMP-1 or LMP-DM, a LMP-1 mutant that fails to induce an antiviral state and to inhibit the lytic replication of EBV (63, 90). The transfected cells were enriched and recovered for several hours before the addition of TPA (see Materials and Methods for details). As shown in Fig. 5A, LMP-1 significantly reduced the expression of K-RTA and K8. Expression of another KSHV lytic gene, vIRF1, was also inhibited (data not shown). Furthermore, LMP-DM, which fails to initiate signals from LMP-1 (94), failed to inhibit KSHV lytic replication, suggesting that the signals derived from LMP-1 are involved in the inhibition. The recombinant adenovirus containing LMP-1 was also used to infect BC3 cells. As shown in Fig. 5B, while the infection by AdGFP still allowed significant lytic replication, the infection of AdLMP-1 inhibited the expression of K-RTA as well as K8. Thus, overexpression of EBV LMP-1 inhibits KSHV lytic replication.

Suppression of LMP-1 enhances KSHV lytic replication.

Having established that overexpression of LMP-1 inhibits KSHV lytic replication, we then examined if endogenous LMP-1 was involved in the lytic replication of KSHV in dually infected PEL cell lines. A small interfering RNA for LMP-1 (siLMP-1) expression plasmid was generated and was able to suppress the expression of LMP-1 significantly in transient-transfection assays with 293T cells (Fig. 6A). siLMP-1 was used to generate a stable cell line with BC1 cells, a PEL line dually infected with both EBV and KSHV, and was able to suppress endogenous LMP-1 expression (Fig. 6B). Sodium butyrate was able to selectively induce the lytic replication of KSHV but not EBV (74). Upon the induction of KSHV lytic replication by sodium butyrate, the siLMP-1-expressing cells had higher expression of K-RTA and K8 than control cells (Fig. 6C). The LMP-1 protein was detectable in several dually infected PEL lines by immunoprecipitation (IP)-Western blot analysis (Fig. 6D). The majority of LMP-1 proteins were lost during the immunoprecipitation process under our experimental condition; the use of IP to show the quantitative differences was unreliable. In addition, immunostaining of LMP-1 and K-RTA in dually infected BC1 cells after treatment with sodium butyrate showed that the majority of cells were either K-RTA or LMP-1 positive, and only a small fraction (<10%) were positive for both (data not shown). All of these data suggested that endogenous LMP-1 was contributing to the control of KSHV lytic replication in dually infected PELs.

Expression of LMP-1 during primary infections.

To address if the interaction between K-RTA and LMP-1 is relevant to the establishment of latencies in dually infected PELs, we examined if LMP-1 is induced during primary infections. First, whether LMP-1 is inducible during KSHV infection of EBV latency cells was examined. 293-EBV cells (KSHV⁻ EBV⁺) were used for the infection by concentrated KSHV. The reason for choosing 293-EBV is that KSHV apparently infects fibroblasts efficiently (49). Given the fact that infection by KSHV of B cells is inefficient (19, 44), infection of 293-EBV cells may be the best available alternative to address alterations in EBV gene expression during primary infection of KSHV. As shown in Fig. 7A, LMP-1 is highly induced by KSHV infection. K-RTA is expressed during primary infection (49). Whether K-RTA is involved in the induction of EBV LMP-1 in primary infection was addressed by the use of siRNA for K-RTA. After the transfection of siRNA for K-RTA into 293-EBV cells, the expression of both LMP-1 and K-RTA was reduced upon KSHV infection, suggesting the involvement of K-RTA in the induction (Fig. 7B).

Second, we examined if LMP-1 is expressed during EBV infection of KSHV latency cells. EBV was used to infect Cro6 (KSHV⁺ EBV⁻) cells. RNA was isolated from infected cells for RT-PCR analyses 24 h later. The infection of KSHV latency cells by EBV was associated with EBV LMP-1 expression (Fig. 7D). Therefore, in both scenarios, LMP-1 is expressed during primary infections.

Induction of lytic replications during primary infection.

EBV latency can be disrupted by several treatments, including viral superinfection (4, 21, 29). To test what may happen in EBV latency, we examined if KSHV could disrupt EBV latency by inducing lytic replication of EBV. EBV EA-D (BMRF-1) expression was used as a marker for lytic replication. The essential function of EA-D in EBV lytic replication has been well established, and use of EA-D as an indicator of lytic replication has been appreciated in the field for years (28, 32, 34, 78-80, 89, 90). KSHV infection of 293-EBV induced EBV EA-D expression (Fig. 7C). Thus, KSHV infection of EBV latency cells induced EBV lytic replication. However, KSHV infection still induced LMP-1 in BZLF1-KO (KSHV⁻ EBV⁺) cells, in which EBV lytic replication could not be induced (27) (Fig. 7C), suggesting that EBV lytic replication plays a limited role in the induction of LMP-1 by KSHV.

Finally, EBV was used to infect KSHV⁺ EBV⁻ PEL cells. Because infection by EBV of Cro6 cells is inefficient (<5%; data not shown), RT-PCR was used to detect the expression of K-RTA expression. As shown in Fig. 7D, infection of Cro6 (KSHV⁺ EBV⁻) cells by EBV induced K-RTA expression. Due to well-established functions of K-RTA, the results also suggested that EBV infection might induce lytic replication of KSHV.