The replication and transcription activator of murine gammaherpesvirus 68 cooperatively enhances cytokine-activated, STAT3-mediated gene expression

Abstract

Gammaherpesviruses (γHVs) have a dynamic strategy for lifelong persistence, involving productive infection, latency, and intermittent reactivation. In latency reservoirs, such as B lymphocytes, γHVs exist as viral episomes and express few viral genes. Although the ability of γHV to reactivate from latency and re-enter the lytic phase is challenging to investigate and control, it is known that the γHV replication and transcription activator (RTA) can promote lytic reactivation. In this study, we provide first evidence that RTA of murine γΗV68 (MHV68) selectively binds and enhances the activity of tyrosine-phosphorylated host STAT3. STAT3 is a transcription factor classically activated by specific tyrosine 705 phosphorylation (pTyr⁷⁰⁵-STAT3) in response to cytokine stimulation. pTyr⁷⁰⁵-STAT3 forms a dimer that avidly binds a consensus target site in the promoters of regulated genes, and our results indicate that RTA cooperatively enhances the ability of pTyr⁷⁰⁵-STAT3 to induce expression of a STAT3-responsive reporter gene. As indicated by coimmunoprecipitation, in latently infected B cells that are stimulated to reactivate MHV68, RTA bound specifically to endogenous pTyr⁷⁰⁵-STAT3. An in vitro binding assay confirmed that RTA selectively recognizes pTyr⁷⁰⁵-STAT3 and indicated that the C-terminal transactivation domain of RTA was required for enhancing STAT3-directed gene expression. The cooperation of these transcription factors may influence both viral and host genes. During MHV68 de novo infection, pTyr⁷⁰⁵-STAT3 promoted the temporal expression of ORF59, a viral replication protein. Our results demonstrate that MHV68 RTA specifically recognizes and recruits activated pTyr⁷⁰⁵-STAT3 during the lytic phase of infection.

Keywords: STAT3; gammaherpesvirus; gene expression; infection; signal transduction; tyrosine; tyrosine phosphorylation; virology.

Figure 1.

MHV68 RTA cooperatively enhances cytokine-activated, STAT3-mediated gene expression. A, induction of the α2-macroglobulin luciferase reporter gene was measured in Hep3B cells expressing STAT3 and/or FLAG-tagged MHV68 RTA (FLAG-RTA) as indicated. Cells were untreated or stimulated with IL-6. Relative -fold induction is shown in cells untreated or stimulated with IL-6. Dark bars denote expression of ectopic STAT3 with FLAG-RTA. Increasing ratio of RTA to STAT3 noted was 0.06, 0.2, and 1.0. Western blot of protein lysates from one representative experiment is shown below graph. B, enhancement of STAT3 transactivation by RTA is dependent on STAT3 tyrosine 705 phosphorylation. The reporter gene assay described in (A) was used with wild-type STAT3 or a site-directed mutation Y705F (Y/F) in Hep3B cells. MHV68 RTA (FLAG-RTA) was added as indicated. C, KSHV-RTA cooperates with host STAT3. V5-tagged RTA from the human Kaposi's sarcoma-associated herpesvirus (V5-K-RTA) was coexpressed with STAT3 as in (A). Increasing ratio of K-RTA to STAT3 was 0.5, 2, and 4. Error bars represent ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test for duplicates of three independent experiments or triplicates of two independent experiments.

Figure 2.

Endogenous STAT3 and MHV68 RTA are binding partners. A, Western blot of FLAG-RTA protein expression induced with doxycycline (DOX) in murine MHV68+ HE-RIT B cells. B, MHV68 viral replication during reactivation in HE-RIT cells measured by quantitative PCR. The -fold increase in viral ORF46 gene normalized to cellular GAPDH gene. Representative experiment is shown; error bars indicate ± S.E. C, Western blot of tyrosine705-phosphorylated STAT3 (pTyr-STAT3), serine727-phosphorylated STAT3 (pSer-STAT3) and total STAT3 (STAT3) in response to IL-6 in HE-RIT cells. D, coimmunoprecipitation of RTA with endogenous STAT3. HE-RIT cells were treated with DOX or IL-6 or both DOX and IL-6, as indicated. FLAG-RTA was collected on anti-FLAG magnetic beads and bound endogenous STAT3 was detected by Western blotting. Western blots shown for bound pTyr-STAT3, STAT3, FLAG-RTA, and β-actin.

Figure 3.

Preferential binding of MHV68 RTA to tyrosine-phosphorylated STAT3 is seen in vitro. A, recombinant MBP and unphosphorylated STAT3 (MBP-U-STAT3) were prepared from BL21 control bacteria. Elk tyrosine kinase expressing bacteria (TKB-1), were used to prepare MBP, MBP-STAT3 Y705F mutant (MBP-Y705F-STAT3), and tyrosine-phosphorylated STAT3 (MBP-pTyr-STAT3). Recombinant proteins were bound to amylose beads and incubated with FLAG-RTA from HE-RIT cell lysates. Western blot analysis detected bound RTA, pTyr STAT3, and STAT3. Bottom panels show recombinant MBP or MBP-STAT3 input detected by Ponceau S staining. B, linear diagram of STAT3 protein with noted functional domains and deletions used in binding assay. MBP-pTyr-STAT3 and N-terminal deletion proteins were bound to amylose and incubated with FLAG-RTA expressed in HEK-293T. Bound RTA was detected by Western blotting (top panel). Input pTyr-STAT3 and total STAT3 are shown.

Figure 4.

The C-terminal domain of MHV68 RTA is required for synergistic induction of a STAT3-responsive gene. A, linear diagram of MHV68 RTA full-length (WT) and deletion mutants Rd1, Rd2, and TAD. Location of DNA-binding domain (DBD) and transcriptional activation domain (TAD) of RTA are indicated. B, induction of α2-macroglobulin luciferase reporter gene by coexpression of STAT3 with empty vector (v) or the indicated RTA constructs in Hep3B cells treated with IL-6. Values are means of triplicate samples in two independent experiments. Error bars represent ± S.E. The enhancement in luciferase activity by WT RTA is significant, p, < 0.0001, using one-way ANOVA followed by Tukey's multiple comparison test. Western blotting of RTA expression is shown in supplemental Fig. 5S. C, evaluation of mutant RTA binding to unphosphorylated STAT3 (U) or tyrosine phosphorylated STAT3 (pTyr) in vitro. Recombinant MBP-U-STAT3 or MBP-pTyr-STAT3 was bound to amylose beads and incubated with FLAG-RTA WT, Rd1, Rd2, TAD, or vector (v) alone expressed in HEK-293T cells. Bound RTA was detected by Western blotting (right panel), and input MBP-STAT3 was evaluated by Coomassie Blue stain (bottom panel).

Figure 5.

IL-6 augments MHV68 lytic gene expression via the STAT3 pathway. A, MHV68 de novo viral lytic replication in Hep3B cells measured by plaque assay. Data are from one representative experiment with error bars ± S.E. B, protein profile of viral gene expression and STAT3 measured by Western blotting with specific antibodies to viral LANA, ORF59, ORF75C, host STAT3, and GAPDH during single step growth curve. C, diagram of viral infection and IL-6 addition. Hep3B cells were serum-starved and infected with MHV68-H2BYFP at m.o.i. 10. Infected cells were treated with IL-6 at 10 h post infection and harvested after 2 h of IL-6 treatment. Western blotting for viral proteins and STAT3 as indicated. D, murine embryonic fibroblasts corresponding to stat3^fl/fl and stat3^−/− were serum-starved and infected with MHV68-H2BYFP at m.o.i. 3 without IL-6 addition and harvested after 12 or 24 h of infection. Levels of viral proteins and host STAT3 and β-actin were detected by Western blotting with specific antibodies.