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J Virol. 2012 February; 86(4): 1930–1941.
PMCID: PMC3302405

Murine Gammaherpesvirus 68 Evades Host Cytokine Production via Replication Transactivator-Induced RelA Degradation

Abstract

Cytokines play crucial roles in curtailing the propagation and spread of pathogens within the host. As obligate pathogens, gammaherpesviruses have evolved a plethora of mechanisms to evade host immune responses. We have previously shown that murine gammaherpesvirus 68 (γHV68) induces the degradation of RelA, an essential subunit of the transcriptionally active NF-κB dimer, to evade cytokine production. Here, we report that the immediately early gene product of γHV68, replication transactivator (RTA), functions as a ubiquitin E3 ligase to promote RelA degradation and abrogate cytokine production. A targeted genomic screen identified that RTA, out of 24 candidates, induces RelA degradation and abolishes NF-κB activation. Biochemical analyses indicated that RTA interacts with RelA and promotes RelA ubiquitination, thereby facilitating RelA degradation. Mutations within a conserved cysteine/histidine-rich, putative E3 ligase domain impaired the ability of RTA to induce RelA ubiquitination and degradation. Moreover, infection by recombinant γHV68 carrying mutations that diminish the E3 ligase activity of RTA resulted in more robust NF-κB activation and cytokine induction than did infection by wild-type γHV68. These findings support the conclusion that γHV68 subverts early NF-κB activation and cytokine production through RTA-induced RelA degradation, uncovering a key function of RTA that antagonizes the intrinsic cytokine production during gammaherpesvirus infection.

INTRODUCTION

Gammaherpesviruses are a group of large DNA viruses that persist within their hosts for life, after primary infection. Members of the subfamily Gammaherpesvirinae include Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), which are causally linked to human malignancies of lymphoid or endothelial/epithelial origin (5, 6). To successfully colonize the host, gammaherpesviruses have evolved various strategies to evade and modulate host innate immune responses (26). Murine gammaherpesvirus 68 (γHV68), a natural pathogen of murid rodents, is closely related to human oncogenic KSHV and EBV (32, 33). γHV68 infection in laboratory strains of mice results in a robust acute infection in the lung and a persistent latent infection in the spleen, thereby offering a tractable small-animal model to interrogate gammaherpesvirus interactions with the host, particularly the immune system.

Innate immunity represents the first line of host defense against invading pathogens. Host pattern recognition receptors (PRRs), including Toll-like receptors, RIG-I-like receptors, and NOD-like receptors, recognize pathogen-associated molecular patterns (PAMPs) and initiate signal transduction, which promotes the synthesis and secretion of inflammatory cytokines and interferons (1, 18, 24). These inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and CCL5 (RANTES), play essential roles in establishing a local antiviral state and recruiting inflammatory cells to the site of infection, thereby mounting a robust immune response. Key to innate immune signal transduction and cytokine production is the activation of two families of transcription factors, i.e., NF-κB and interferon regulatory factors (IRFs). In resting cells, NF-κB dimers are kept latent in the cytosol by the inhibitors of NF-κB, IκBs. Upon an association between PRRs and cognate PAMPs, IκBs (such as IκBα) are phosphorylated by innate immune kinases, e.g., IκB kinase α (IKKα) and IKKβ, and undergo ubiquitination and proteasome-mediated degradation (9, 25). Unleashed NF-κB dimers translocate into the nucleus to upregulate the transcription of genes of diverse immune functions, including cytokines. Thus, NF-κB activation is one of the most critical checkpoints in the regulation of host immune activation and inflammation to combat invading pathogens (2).

The NF-κB transcription factors are also known as Rel homology domain (RHD)-containing proteins and form two categories according to their functions in transcriptional activation. RelA (also known as p65), RelB, and c-Rel are transcriptionally active due to the carboxyl-terminal transcription activation domain (TAD). In contrast, p50 (processed from p110) and p52 (processed from p105) lack the TAD and are transcriptionally inactive (30, 37). These subunits can form hetero- and homodimers that differentially regulate gene transcription. Specifically, transcriptional activation is dependent on one of the three TAD-containing RHD proteins, whereas dimers constituting the transcriptionally inactive p50 and p52 have been postulated to inhibit NF-κB activation (30, 37). Among the NF-κB subunits, RelA is the most ubiquitously and abundantly expressed, highlighting its critical role in the signal transduction of fundamental biological processes, such as immune responses. Indeed, the regulation of NF-κB activation has been investigated primarily by using RelA as the prototype NF-κB subunit. Although the regulated degradation of IκBα remains one of the best-defined mechanisms governing RelA activation, recent studies also shed light on posttranslational modifications of the RelA subunit in tuning NF-κB activation, including phosphorylation and acetylation (8, 27). In particular, RelA phosphorylation within its TAD was discovered to endow a distinct fate of this key transcription factor, e.g., activation or degradation, in a site-specific manner. However, the physiological cues that determine the site-specific phosphorylation and the outcome thereof remain unknown.

We previously reported that γHV68 activates the mitochondrion antiviral signaling (MAVS)-IKKβ pathway to promote viral transcriptional activation and lytic infection (10). Moreover, MAVS and IKKβ are necessary for γHV68 to abrogate NF-κB activation and cytokine production via induced RelA degradation (12). In this study, we identified the replication transactivator (RTA), encoded by the immediate-early gene ORF50, as a viral effector that abrogates NF-κB activation during early γHV68 infection. Mechanistically, γHV68 RTA promotes the ubiquitination and degradation of RelA. Mutations targeting conserved residues of the E3 ligase domain of RTA impaired RelA ubiquitination and degradation induced by RTA. Infection with recombinant γHV68 carrying these mutations triggered more robust cytokine production than did infection with wild-type γHV68. These findings provide us the first insight into the in vivo roles of the E3 ligase activity of RTA in gammaherpesvirus infection.

MATERIALS AND METHODS

Plasmids.

For protein expression in mammalian cells, MAVS, MyD88, TRAF6, NOD2, Rip2, IKKβ, RelA, enhanced green fluorescent protein (eGFP)-tagged RelA, Flag-tagged wild-type RTA, or an RTA mutant carrying Cys141Ser and Cys152Ser mutations (designated RTA C/S) was cloned into pcDNA5/FRT/TO (Invitrogen). The γHV68 expression library, consisting of plasmids containing individual γHV68 open reading frames (ORFs), was constructed by using pEF1/V5-His (Invitrogen), and protein expression was confirmed by immunoblotting.

Cells and viruses.

NIH 3T3 cells, mouse embryonic fibroblasts (MEFs), 293T cells, and T-REx 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Mediatech) containing 8% newborn calf serum (NCS), fetal bovine serum (FBS), and tetracycline-free FBS, respectively. Wild-type γHV68, γHV68.K3/GFP, and recombinant γHV68 were amplified in NIH 3T3 cells, and viral titers were determined by a plaque assay using NIH 3T3 cell monolayers.

Generation of recombinant γHV68.

The bacterial artificial chromosome (BAC) system was used to generate recombinant γHV68 as previously described (10, 11). Briefly, wild-type RTA and the RTA C/S allele were PCR amplified with overlapping PCR primers. Purified PCR products, along with RTA null BAC clone 8-42 (31), containing a transposon within the N terminus of RTA (between nucleotide 68075 and 68076, according to NCBI accession number U97553), were transfected into 293T cells with Lipofectamine 2000 (Invitrogen). Virus in the supernatant was amplified with NIH 3T3 cells. To isolate circular BAC DNA, NIH 3T3 cell monolayers were infected with recombinant γHV68, and BAC DNA was extracted according to Hirt's protocol (3, 17) and electroporated into ElectroMAX DH10B cells (Invitrogen). To rule out an unexpected chromosome rearrangement, BAC DNA containing the γHV68 genome was extracted, digested with KpnI and AflII, and resolved on a 0.8% agarose gel. To confirm the desired mutations at cysteines 141 and 152 of RTA, the RTA allele was amplified by PCR and sequenced. Validated BAC clones were transfected into NIH 3T3 cells, and recombinant γHV68 was further amplified in NIH 3T3 cells. To ensure that equivalent amounts of infectious virions of recombinant viruses were used in subsequent experiments, a MEF monolayer was infected with serially diluted recombinant γHV68 carrying wild-type RTA (γHV68.NR) or the RTA C/S allele (γHV68.C/S) by centrifugation at a relative centrifugal force (RCF) of 300 at 37°C for 1 h. Total genomic DNA was extracted from MEFs at 2 h postinfection (p.i.), and viral genomes were assessed by quantitative real-time PCR.

Plaque assay.

To examine the ex vivo growth kinetics of recombinant γHV68, NIH 3T3 cells were infected with equivalent numbers of infectious virions of γHV68.NR and γHV68.C/S at a multiplicity of infection (MOI) of 0.01 or 5 (γHV68.NR). The viral titer of cell lysates was assessed by a plaque assay on NIH 3T3 monolayers as previously described (10, 11). Briefly, after three rounds of freezing and thawing, 10-fold serially diluted whole-cell lysates (WCLs) were added onto NIH 3T3 monolayers and incubated for 2 h at 37°C. DMEM containing 2% NCS and 0.75% methylcellulose (Sigma) was then added after the removal of the supernatant. Plaques were counted at 6 days p.i. The detection limit for this assay was 5 PFU.

Mice and viral infections.

Gender-matched, 6- to 8-week-old C57BL/6 (B6) littermate mice were obtained from the Animal Resource Center, University of Texas Southwestern Medical Center. To assess cytokine production by recombinant γHV68, B6 mice (eight mice per group) were inoculated intranasally with 1 × 105 PFU of γHV68.NR and γHV68.RTA C/S. Lungs were collected at the indicated time points and homogenized in DMEM. All mice were housed in the animal facility of the University of Texas Southwestern Medical Center, and all experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Southwestern Medical Center.

RT-PCR and qRT-PCR.

To determine the relative levels of host cytokine and viral transcripts, reverse transcription (RT)-PCR and quantitative real-time PCR (qRT-PCR) were performed as previously described (10, 12). Briefly, total RNA was extracted from NIH 3T3 cells or mouse lungs by using TRIzol reagent (Invitrogen). To remove genomic DNA, total RNA was digested with RNase-free DNase I (New England Biolabs) at 37°C for 1 h. DNase I digestion was quenched by heat inactivation at 70°C for 20 min, and total RNA was repurified with TRIzol reagent. cDNA was prepared with 1.5 μg total RNA, reverse transcriptase (Invitrogen), and 0.5 μg/μl primer oligo(dT)12-19. RNA was then removed by incubation with RNase H (Epicentre). The abundance of cytokine mRNAs and viral transcripts was assessed by qRT-PCR using a 7500 Fast real-time PCR system (Applied Biosystems). Mouse β-actin was used as an internal control. To assess viral genomic DNA in recombinant γHV68-infected MEFs, total genomic DNA was purified by phenol-chloroform extraction and ethanol-ammonia acetate precipitation after digestion with proteinase K (Qiagen). The abundance of the γHV68 genome in 20 ng total genomic DNA was assessed by real-time PCR using primers specific for RTA and ORF60. All primers were synthesized by Invitrogen and validated individually (Table 1).

Table 1
Primers used for quantitative real-time PCR

Luciferase reporter assay.

293T cells were seeded into 24-well plates (1 × 105 cells/cm2) at 16 h before transfection. A total of 500 ng of plasmid cocktail per well was transfected by the calcium phosphate method. To identify the viral protein(s) that induced RelA degradation, we transfected 293T cells with a plasmid cocktail that includes 100 ng of NF-κB luciferase reporter plasmid (3κBp-luc), 200 ng of pCMV–β-galactosidase plasmid (β-Gal), 50 ng of RelA plasmid, and 50 ng of pEF1/V5-His harboring γHV68 genes. To examine the inhibitory effect of RTA on NF-κB activation, we transfected 293T cells with a plasmid cocktail consisting of 100 ng of 3κBp-luc, 200 ng of β-Gal, 100 ng of pcDNA5/FRT/TO containing wild-type RTA or the RTA C/S variant, and 100 ng of pcDNA5/FRT/TO expressing RelA, MAVS, MyD88, TRAF6, NOD2, Rip2, or IKKβ. At 20 h posttransfection, the firefly luciferase activity and β-galactosidase activity in whole-cell lysates were assessed with FLUOstar Omega (BMG Labtech).

IP and immunoblotting.

For immunoprecipitation (IP) and immunoblot assays, cells were harvested, rinsed once with ice-cold phosphate-buffered saline (PBS), and resuspended with NP-40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 5 mM EDTA) or radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, 5 mM EDTA) supplemented with a protease inhibitor cocktail (Roche). The centrifuged supernatant was precleared with protein A/G-agarose at 4°C for 1 h and subjected to precipitation by incubation with anti-RelA antibody and protein A/G-agarose or anti-Flag M2-conjugated agarose. Precipitated proteins were washed extensively with NP-40 buffer or RIPA buffer and eluted with 1× SDS-PAGE loading buffer by boiling at 95°C for 10 min. For immunoblot analysis, WCLs (20 μg) or precipitated proteins were resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking of the membrane with 3% milk, immunoblot detection was performed with the corresponding primary antibodies by incubation at 4°C overnight and with secondary peroxidase-conjugated antibody for 60 min at room temperature. Proteins were visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and the LAS-3000 imaging system (FujiFilm).

Pulse-chase analysis of protein half-life.

To assess the half-life of RelA with and without γHV68 RTA, 293T cells were transiently transfected with plasmids carrying RelA, wild-type RTA, or the RTA C/S allele and pulse-labeled with [35S]methionine-cysteine ([35S]Met-Cys) for 30 min. After extensive washing with PBS, cells were chased with normal medium up to 4 h. At various time points, cells were harvested, washed with cold PBS, resuspended in RIPA buffer, and lysed by passage through 26-gauge syringe needles 15 times. Centrifuged supernatants were precleared with protein A/G-agarose and incubated with anti-RelA polyclonal antibody and protein A/G-agarose at 4°C for 4 h. Precipitated proteins were washed extensively with RIPA buffer and analyzed by SDS-PAGE and autoradiography.

EMSA.

An electrophoresis mobility shift assay (EMSA) was performed as previously described (12). NIH 3T3 cells were infected with γHV68.RTA or γHV68.RTA C/S (MOI = 5) and harvested at the indicated time points. Cells were washed once with ice-cold PBS, scraped into 5 ml cold PBS on ice, and centrifuged at 2,000 × g at 4°C for 5 min. The cell pellet was resuspended in ice-cold hypotonic lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, 0.65% Nonidet P-40). Nuclei were spun down and rinsed with ice-cold hypotonic lysis buffer without Nonidet P-40. Nuclei were resuspended in a low-salt buffer (20 mM HEPES [pH 7.9], 2 mM EDTA [pH 8.0], 20 mM KCl, 1.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 25% glycerol). A high-salt buffer (20 mM HEPES [pH 7.9], 2 mM EDTA [pH 8.0], 800 mM KCl, 1.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 25% glycerol) was then added in a dropwise fashion with gentle stirring. The supernatant (nuclear extract) was collected by centrifugation at 25,000 × g for 30 min at 4°C.

Nuclear extracts were analyzed for NF-κB activation by EMSA. Two micrograms of nuclear extracts was incubated with a 32P-labeled oligonucleotide (Promega) containing the NF-κB consensus site (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) for 15 min at room temperature in a binding reaction mixture containing 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 50 mM NaCl, 1 mM MgCl2, 0.5 mM dithiothreitol, 0.05 mg/ml poly(dI-dC) (Sigma), and 4% glycerol. DNA-protein complexes were subjected to electrophoresis on 6% native polyacrylamide gels (0.25× Tris-borate-EDTA [TBE]) at a constant current of 9 mA. Gels were dried and analyzed by use of a Storm 820 instrument (Amersham Bioscience) for autoradiography.

Antibodies.

Commercial antibodies used in this study include mouse anti-Flag (Sigma), mouse anti-green fluorescent protein (GFP) (Covance), mouse anti-β-actin (Abcam), mouse anti-ubiquitin (P4D1) (Santa Cruz Biotech), rabbit anti-RelA (C-20) (Santa Cruz Biotech), and rabbit anti-glutathione S-transferase (GST) (Santa Cruz Biotech) antibodies. To generate antibody to γHV68 RTA, the GST fusion protein containing the RTA C terminus (amino acids [aa] 443 to 589) was used to immunize rabbits. The specificity of rabbit polyclonal antibody to RTA was examined against preimmune sera.

Statistical analysis.

The statistical significance (P value) was calculated by unpaired two-tailed Student's t test. A P value of <0.05 was considered statistically significant.

RESULTS

γHV68 RTA subverts NF-κB activation.

We recently reported that γHV68 hijacks the MAVS adaptor and its immediately downstream IKKβ kinase to abrogate NF-κB activation by inducing the degradation of RelA, a key subunit of the transcriptionally active NF-κB dimers (12). Additionally, RelA degradation induced by γHV68 is transient and occurs during early stages of infection (2 to 4 h postinfection), suggesting that viral structural components or immediately early gene products are responsible for RelA degradation. To identify the viral protein(s) that induces RelA degradation, we employed an NF-κB luciferase reporter assay as a surrogate to screen for a viral factor(s) that is sufficient to inhibit NF-κB activation. Given that γHV68 targets RelA to terminate NF-κB activation, we expressed exogenous RelA to activate NF-κB. To facilitate the functional screen, we cloned most (72 out 80) of the putative open reading frames (ORFs) of γHV68 into a mammalian expression plasmid, and their expressions were validated by immunoblotting (data not shown). This experiment identified RTA (ORF50) as the only γHV68 factor, out of 24 candidate viral proteins that include structural components and immediately early gene products, which potently inhibited RelA-mediated NF-κB activation (Fig. 1A). Moreover, the coexpression of RTA drastically abolished NF-κB activation induced by upstream components of the NF-κB pathway, including the MAVS adaptor and the IKKβ kinase (Fig. 1B and C). This result supports the premise that RTA targets RelA to prevent NF-κB activation. To examine whether the physiologically relevant level of γHV68 RTA is sufficient to inhibit NF-κB activation, we employed T-REx 293 stable cell lines that express RTA under the control of doxycycline, a functional analog of tetracycline. A luciferase reporter assay revealed that TRAF6-induced NF-κB activation was dramatically reduced by the tetracycline-inducible expression of RTA (Fig. 1D). The RTA induction by doxycycline treatment was confirmed by quantitative real-time PCR (Fig. 1E). Finally, doxycycline-induced RTA expression sufficiently abrogated the upregulation of TNF-α and CXCL1 mRNAs induced by Sendai virus infection (Fig. 1F). These results collectively identified γHV68 RTA that targets RelA to abrogate NF-κB activation.

Fig 1
Identification of γHV68 RTA that abrogates NF-κB activation. (A) 293T cells were transfected with a reporter plasmid cocktail and a plasmid containing the individual γHV68 gene. NF-κB activation by RelA was assessed by ...

γHV68 RTA induces RelA ubiquitination and degradation.

To dissect the molecular mechanism of the RTA-mediated inhibition of RelA-dependent NF-κB activation, we expressed eGFP-tagged RelA and γHV68 RTA in 293T cells. The results of fluorescence microscopy showed that the coexpression of RTA greatly reduced the expression level of the eGFP-RelA protein but not that of eGFP (Fig. 2A), suggesting that γHV68 RTA specifically targets RelA for inhibition. Indeed, immunoblot analysis revealed that RTA coexpression diminished RelA protein levels and that RelA protein levels were restored by treatment with MG132, a proteasome inhibitor, but not by treatment with chloroquine, a lysosome inhibitor (Fig. 2B). This result implies that proteasome-mediated degradation underpins the RTA-dependent RelA inhibition. To determine whether RTA induces RelA ubiquitination to facilitate RelA degradation, we examined RelA ubiquitination by IP, with or without RTA expression. Indeed, RelA-ubiquitinated species were observed only in cells that expressed RTA and were treated with MG132 (Fig. 2C). The fact that ubiquitinated RelA was observed only in the presence of MG132 implies that RelA ubiquitination by RTA is the rate-limiting step of RelA degradation. Finally, we determined the half-life of RelA by a pulse-chase assay. In agreement with RTA promoting RelA degradation, we discovered that RTA expression reduced the half-life of RelA to approximately 1.5 h in 293T cells, whereas the half-life of RelA is approximately 6 h without RTA expression (Fig. 2D).

Fig 2
γHV68 RTA promotes ubiquitination and degradation of RelA. (A) 293T cells were transfected with plasmids containing RTA and eGFP-tagged RelA or eGFP. eGFP- and eGFP-RelA-expressing cells were visualized by fluorescence microscopy (left) and Nomarski ...

The observation that RTA targets RelA for degradation suggests that RTA may physically interact with RelA. To test this hypothesis, we cotransfected 293T cells with plasmids expressing Flag-tagged RTA and eGFP-tagged RelA and performed co-IP using whole-cell lysates of transfected 293T cells. The precipitation of RTA efficiently recovered RelA by immunoblot analysis (Fig. 2E). It is noteworthy that the coexpression of RTA greatly reduced RelA protein levels in transfected cells, consistent with the finding that RTA diminishes RelA protein expression (Fig. 2A and B). Moreover, the interaction between RelA and RTA was further validated by reverse co-IP and the finding that MG132 treatment enhanced the RelA-RTA interaction (Fig. 2F). To further examine whether RTA interacts with RelA during γHV68 infection, we infected mouse embryonic fibroblasts (MEFs) and performed co-IP using an anti-RelA antibody. We observed that RTA was readily detected within protein complexes that were precipitated with rabbit anti-RelA antibody but not with a control rabbit antibody (Fig. 2G). Taken together, we demonstrated that RTA physically interacts with RelA and induces the rapid degradation of RelA via the ubiquitin/proteasome pathway.

A putative E3 ligase domain is necessary for γHV68 RTA to induce RelA ubiquitination and degradation.

The fact that RTA interacts with RelA and increases RelA ubiquitination suggests that RTA either functions as a bona fide ubiquitin E3 ligase or engages a cellular ubiquitin E3 ligase(s) to promote RelA degradation. The closely related KSHV RTA was shown previously to act as a functional ubiquitin E3 ligase and promote the polyubiquitination of IRF7 (41). Sequence alignment revealed that γHV68 RTA and KSHV RTA share the ubiquitin E3 ligase domain, a conserved cysteine-rich domain (Fig. 3A). To assess the role of the putative ubiquitin E3 ligase domain in γHV68 RTA, we mutated two highly conserved cysteine residues (cysteine 141 and 152) to serines, designated RTA C/S, and examined the ability of the RTA C/S variant to induce RelA ubiquitination and degradation. In the presence of MG132, the RTA C/S variant failed to increase RelA ubiquitination compared to wild-type RTA (Fig. 3B), indicating that the two cysteines are critical for the RTA-induced ubiquitination of RelA. As KSHV RTA was shown to undergo auto-ubiquitination, we examined whether the cysteine-rich domain is important for γHV68 RTA ubiquitination in transfected 293T cells. Indeed, a high-molecular-weight species of RTA was observed as “smearing” when precipitated RTA was analyzed by immunoblotting for ubiquitin, which is indicative of RTA ubiquitination (Fig. 3C). Interestingly, we noted that significantly higher levels of ubiquitinated RTA C/S than of wild-type RTA were detected in transfected cells, particularly when cells were treated with MG132 (Fig. 3C). This result suggests that the cysteine-rich domain is not necessary for RTA ubiquitination; rather, it is required for RTA-mediated RelA ubiquitination.

Fig 3
A conserved ubiquitin E3 ligase activity is necessary for γHV68 RTA to induce RelA ubiquitination and degradation. (A) The ubiquitin E3 ligase domain is highly conserved between γHV68 RTA and KSHV RTA. Two solid triangles mark the cysteine ...

To further characterize RTA-inhibited RelA protein expression, we carried out pulse-chase experiments. Consistent with the reduced RelA ubiquitination induced by the RTA C/S variant, we found that the RTA C/S variant reduced the half-life of the RelA protein to 4 h in 293T cells, whereas wild-type RTA reduced the half-life of the RelA protein from approximately 6 h to 1.5 h (Fig. 3D). It is important that the RTA C/S variant is expressed at levels 2.5-fold higher than the level of wild-type RTA (Fig. 3E), implying that the effect of the two cysteine-to-serine mutations is underestimated. To determine whether the two cysteine-to-serine mutations disrupt the interaction between RTA and RelA, we performed co-IP using transfected 293T cells. In fact, the precipitation of the RTA C/S variant recovered more RelA than the precipitation of wild-type RTA, consistent with the finding that MG132 treatment enhanced the RelA-RTA interaction by “arresting” the protein complex (Fig. 3F). This result supports the conclusion that the two cysteine-to-serine mutations do not impede the interaction of RTA with RelA.

To further assess the effect of the RTA C/S variant on RelA protein levels, we coexpressed RelA with either wild-type RTA or the RTA C/S variant in 293T cells. Remarkably, wild-type RTA, despite being expressed at 10% of the levels of the RTA C/S variant, reduced RelA protein levels by 22% more than did the RTA C/S variant (Fig. 4A, lanes 3 and 4). Treatment with MG132 also increased RelA protein levels when the RTA C/S variant was coexpressed, suggesting that the RTA C/S variant contains residual activity to promote RelA degradation (Fig. 4A, compare lanes 5 and 6 to lanes 3 and 4). Moreover, a further increase in the level of the RTA C/S variant by 2-fold had no apparent effect on the RelA protein level (Fig. 4A, lane 7).

Fig 4
Reduced ability of the RTA C/S variant to inhibit NF-κB activation. (A) 293T cells were transfected with plasmids containing RelA and wild-type RTA (WT) or the RTA C/S variant (C/S). At 20 h posttransfection, cells were treated with MG132 (20 ...

To quantify the ability of the RTA C/S variant to terminate NF-κB activation, we examined RelA- and TRAF6-induced NF-κB activation by a luciferase reporter assay when RelA or TRAF6 was coexpressed with wild-type RTA or the RTA C/S variant. We found that wild-type RTA was approximately 8- to 10-fold more potent in inhibiting RelA-induced NF-κB activation than the RTA C/S variant (Fig. 4B). Collectively, these results demonstrated that the conserved cysteine-rich domain is necessary for RelA ubiquitination and degradation induced by γHV68 RTA.

Generation of recombinant γHV68 carrying mutations that interrupt the RTA ubiquitin E3 ligase domain.

Given that the Cys-to-Ser mutations greatly impaired the ability of RTA to induce RelA degradation, we reasoned that recombinant γHV68 carrying the RTA C/S allele (γHV68.C/S) inhibits NF-κB activation less effectively than γHV68 carrying wild-type RTA (γHV68.NR), thereby inducing more robust cytokine production than that of wild-type γHV68. To test this hypothesis, we first generated recombinant γHV68.NR and γHV68.C/S. Taking advantage of the bacterial artificial chromosome (BAC) containing the γHV68 genome with a transposon insertion that inactivates RTA (RTA null) (31), we have developed an efficient approach to obtain recombinant γHV68 containing the desired mutations, e.g., RTA C/S (Fig. 5A) (10, 13). This approach utilizes the homologous recombination that occurs between BAC DNA and PCR products carrying engineered mutations. To exclude potential large DNA rearrangements, we performed DNA digestion with two restriction endonucleases, KpnI and AflII, followed by 0.8% agarose gel electrophoresis. The removal of the kanamycin cassette within the ORF50 locus reduced a 9.1-kb fragment to a 7.8-kb counterpart released by KpnI digestion (Fig. 5B) and reduced a 4.2-kb fragment to a 2.9-kb counterpart released by AflII digestion (Fig. 5C). Furthermore, there was no additional change when the patterns of the three digested BAC DNAs were compared, suggesting that no detectable DNA chromosome rearrangement exists in the genomes of recombinant γHV68. Additionally, mutations of the RTA C/S allele were confirmed by DNA sequencing using the PCR product amplified from recombinant viral genomic DNA.

Fig 5
Generation and characterization of recombinant γHV68 carrying the RTA C/S mutant. (A) γHV68 carrying wild-type RTA or the RTA C/S variant was generated via a recombination-based strategy. Cm, chloramphenicol; Kan, kanamycin. (B and C) ...

To assess the effect of the C/S mutations on γHV68 lytic replication, we examined the growth kinetics of two recombinant γHV68 viruses (γHV68.NR and γHV68.C/S) on highly permissive NIH 3T3 cells at MOIs of 0.01 and 2. To ensure that equivalent amounts of recombinant γHV68 were used to infect NIH 3T3 cells, we normalized the viral input with intracellular viral genomes by quantitative real-time PCR (qRT-PCR). With infection at an MOI of 5 (with γHV68.NR), γHV68.C/S exhibited delayed replication within the first day postinfection compared to γHV68.NR (Fig. 5D). From 2 days p.i., γHV68.C/S replicated to levels similar to those of wild-type γHV68.NR (Fig. 5D), which is indicative of an equivalent replication capacity at a high MOI. In contrast, at an MOI of 0.01, recombinant γHV68.C/S demonstrated delayed replication and reached its maximal yield 1 log lower than that of γHV68.NR (Fig. 5E), likely due to reduced replication initiation. Collectively, these results support the conclusion that the C/S mutations do not impair γHV68 lytic replication ex vivo.

More robust cytokine production is induced by recombinant γHV68.C/S than by wild-type γHV68.NR.

To evaluate the contribution of RTA to evading NF-κB activation, we assessed NF-κB activation after infection with recombinant γHV68. To examine whether RTA E3 ligase activity is necessary for RelA degradation, we infected NIH 3T3 cells with recombinant γHV68 at an MOI of 20. Notably, γHV68 infection at an MOI of 5 is sufficient to terminate NF-κB activation, but infection at an MOI of 20 or higher is necessary to observe RelA degradation, presumably to activate the entire pool of RelA and maximize the number of RTA molecules. As shown in Fig. 6A, γHV68.NR, but not the γHV68.C/S mutant, diminished RelA protein levels at 4 h p.i. This result shows the in vivo biological significance of the RTA E3 ligase activity toward RelA defined ex vivo. We noted that the RelA degradation induced by γHV68 occurs early during viral infection, within 2 to 4 h postinfection in MEFs, and that the replication kinetics of γHV68 progress more rapidly in NIH 3T3 cells than in MEFs. Thus, we focused on NF-κB activation within the first 2 h postinfection in γHV68-infected NIH 3T3 cells. We initially examined NF-κB activation with an antibody specific for the serine 536-phosphorylated form of RelA (RelA.S536p), a marker for activated RelA that recruits p300 for transcriptional activation (27). While γHV68.NR infection induced a gradual increase in the RelA.S536p level, γHV68.C/S triggered a more rapid accumulation of RelA.S536p within 30 min postinfection (Fig. 6B). Moreover, we observed an increase in the DNA-binding activity of NF-κB probes in nuclear extracts from cells infected with γHV68.C/S, particularly at 0.5 h p.i., than in nuclear extracts from cells infected with γHV68.NR (Fig. 6C). These results collectively demonstrated that recombinant γHV68.C/S induces earlier NF-κB activation than does wild-type γHV68.NR.

Fig 6
NF-κB activation and cytokine production induced by γHV68.C/S ex vivo. (A) Mouse embryonic fibroblasts (MEFs) were infected with γHV68.NR and γHV68.C/S at an MOI of 20. Whole-cell lysates (WCLs) were prepared at the indicated ...

Next, we examined whether recombinant γHV68.C/S induces more cytokine production than γHV68.NR. We infected NIH 3T3 cells with equal numbers of infectious virions at an MOI of 5 (γHV68.NR) and analyzed cytokine mRNA levels by quantitative real-time PCR. Although γHV68.C/S and γHV68.NR induced similar peak levels of IL-6 and CXCL2 transcripts, the temporal inductions of IL-6 and CXCL2 by γHV68.C/S were shifted 2 h earlier than those induced by γHV68.NR (Fig. 6D), in agreement with the earlier NF-κB activation induced by γHV68.C/S. Remarkably, γHV68.C/S infection robustly elevated the mRNA levels of TNF-α, alpha interferon (IFN-α), and IFN-β at 8 h p.i., whereas γHV68.NR only marginally increased the levels of these mRNAs (Fig. 6D). To determine whether the robust cytokine production in γHV68.C/S-infected cells was due to a lower expression level of the RTA C/S variant in NIH 3T3 cells, we employed quantitative real-time PCR to assess the relative mRNA levels of RTA, which also serves as an indicator of de novo γHV68 lytic replication. Surprisingly, γHV68.C/S had about 5- to 10-fold more RTA transcripts than did γHV68.NR in infected NIH 3T3 cells. This result indicates that an RTA mutant devoid of ubiquitin E3 ligase activity is not able to effectively inhibit cytokine production, despite expression at a higher level than that of wild-type RTA. Taken together, we conclude that the ubiquitin E3 ligase activity of RTA is necessary for γHV68 to antagonize the cytokine response during lytic replication.

To assess the roles of the ubiquitin E3 ligase activity of RTA during γHV68 infection in vivo, we infected BL/6 littermate mice intranasally with 1 × 104 PFU per mouse of γHV68.NR or equivalent infectious virions of γHV68.C/S. Lung cytokines were determined by an enzyme-linked immunosorbent assay (ELISA) at 4 and 7 days p.i. Evidently, γHV68.C/S induced significantly higher levels of CCL5, CXCL1, and TNF-α in the lung than did γHV68.NR at 4 days p.i. (Fig. 7A). At 7 days p.i., infection by γHV68.NR triggered a much more robust production of TNF-α, CXCL1, and IL-6 and slightly more CCL5 (Fig. 7A) than did infection by γHV68.C/S. This result suggests that sustained lytic replication is necessary for cytokine production during late stages of acute infection (e.g., 7 days p.i.) and imply that early cytokine production induced by γHV68.C/S cripples viral lytic replication in vivo. Indeed, a plaque assay revealed that γHV68.NR infection leads to a viral load of approximately 5,000 PFU/lung, whereas the γHV68.C/S load was 2 orders of magnitude lower than that of wild-type γHV68.NR at 4 days p.i. (Fig. 7B). At 7 days p.i., the γHV68.C/S load was under the detection limit, while the γHV68.NR load remained high in the lung. These results exclude the possibility that the more robust cytokine production induced by γHV68.C/S is due to increased viral lytic replication and rather support the conclusion that the failure of the RTA C/S variant to terminate NF-κB activation results in more robust and/or earlier cytokine production during γHV68 infection. We conclude that γHV68.C/S induces earlier cytokine production than wild-type γHV68.NR, which leads to reduced viral lytic replication. Our findings highlight the roles of the E3 ubiquitin ligase activity of RTA in evading cytokine production in vivo.

Fig 7
γHV68.C/S infection results in earlier robust cytokine production than with γHV68.NR infection in vivo. Age- and gender-matched BL/6 littermate mice were infected with 1 × 104 PFU of γHV68.NR or equivalent infectious virions ...

DISCUSSION

In response to viral infection, host PRRs sense pathogen structural components and/or replication intermediates to activate innate immune signaling cascades that culminate in cytokine production. Key to antiviral cytokine production is the NF-κB transcription factors, and RelA is the most critical subunit of the transcriptionally active NF-κB. To persist within their host, viruses, particularly those with large genomes, dedicate a significant portion of their genetic material to the evasion of cytokine production. We have recently reported that γHV68 hijacks MAVS and IKKβ to abrogate NF-κB activation and antiviral cytokine production (12). In this study, we identified γHV68 RTA as a factor that is sufficient to prevent NF-κB activation in transfected cells and necessary to delay cytokine production during early γHV68 lytic replication. Our biochemical analyses demonstrated that RTA interacts with and targets RelA for ubiquitin/proteasome-dependent degradation. Moreover, infection by recombinant γHV68 carrying mutations that abolish the ability of RTA to induce RelA degradation triggered more robust and/or earlier cytokine gene expression. With a low dose of γHV68 (40 PFU) via intranasal infection, the viral load of recombinant γHV68.C/S at 4 and 7 days p.i. was under the detection limit by a plaque assay (our unpublished data). This result suggests that the mutations abolishing the E3 ligase activity of RTA render γHV68 susceptible to host innate immune responses, such as cytokines. Indeed, when mice were infected with 1 × 104 PFU, recombinant γHV68.C/S also induced earlier cytokine production, whereas wild-type γHV68.NR induced higher cytokine levels in the lung at a later time point (7 days p.i.). Conversely, the viral load of recombinant γHV68.C/S was lower than that of wild-type γHV68.NR. Consistent with this finding, the RTA C/S transcript is much more abundant than that of wild-type RTA in NIH 3T3 cells infected with recombinant γHV68, further supporting the conclusion that the E3 ligase activity of RTA is responsible for terminating NF-κB activation. In stark contrast, the γHV68.C/S mutant replicates indistinguishably from or comparably to γHV68.NR in NIH 3T3 cells. Conceivably, antiviral cytokines evaded by RTA-mediated NF-κB termination are key players in curtailing γHV68 replication in vivo. In fact, γHV68.C/S induced a more robust secretion of CCL5 and CXCL1, which can recruit immune cells (e.g., macrophages). Thus, these findings collectively argue that RTA is necessary to prevent NF-κB activation and evades antiviral cytokine production during early acute γHV68 infection. Although KSHV RTA was reported previously to induce the degradation of IRF3 and IRF7 to prevent interferon production (41), our study uncovered the in vivo roles of the E3 ligase activity of RTA in infection by gammaherpesviruses.

It was reported previously that γHV68 and KSHV LANA (ORF73), an essential latent gene product, induce nuclear RelA degradation and inhibit NF-κB activation (20, 28). However, in our reporter assay-based screen, γHV68 LANA failed to significantly inhibit RelA-mediated NF-κB activation, presumably due to the low dose of plasmid (50 ng) that we used. Nevertheless, the LANA-mediated inhibitory effect on NF-κB activation was dispensable for acute viral infection in the lung, although it was essential for latent viral infection in germinal center lymphoid cells (28). Collectively, these studies bolster the conclusion that RTA is a key player in subverting NF-κB activation and cytokine production during acute γHV68 infection.

It is important that NF-κB activation, e.g., induced by RelA overexpression, inhibits RTA-mediated transcription activation and γHV68 lytic replication. High levels of NF-κB activation were postulated previously to facilitate and to represent a key determinant for maintaining γHV68 latent infection. Indeed, latent viral proteins of gammaherpesviruses, including EBV LMP1 (16) and KSHV vFLIP/K13 (7, 23), potently induce NF-κB activation. Additionally, B lymphocytes, a common natural reservoir of gammaherpesviruses, have high levels of intrinsic NF-κB transcriptional activity. These observations support the notion that NF-κB activation is a key determinant of the suppression of gammaherpesvirus lytic replication. However, it was largely unclear how γHV68 manipulates the NF-κB pathway during lytic replication. It came as a surprise that recombinant γHV68 expressing the NF-κB superrepressor in its lytic phase exhibited no significant alteration of either acute infection in the lung or lytic replication in cultured fibroblasts (19). Paradoxically, we previously reported that γHV68 hijacks the MAVS adaptor and IKKβ kinase, signaling molecules that instigate NF-κB activation in response to viral infection, to promote γHV68 lytic replication. It is possible that γHV68 has evolved an effective strategy to antagonize NF-κB activation during lytic replication. In line with this, we recently discovered that γHV68 infection results in rapid RelA degradation in a MAVS- and IKKβ-dependent but IκBα-independent manner (12), providing a plausible explanation for the apparent paradox. These results argue that RTA-induced RelA degradation and NF-κB termination are critical for the efficient lytic replication of γHV68. In support of this conclusion, it was reported previously that RelA inhibits the transcriptional activity of KSHV RTA and γHV68 RTA and that these RTA molecules overcome RelA's inhibition at higher doses in transfected 293T cells (4). Taken together, we conclude that γHV68 RTA overcomes two intrinsic antiviral activities of RelA, i.e., host cytokine gene transcription activation and viral lytic gene transcription inhibition. Retrospectively, our previous reports that γHV68 exploits the MAVS-IKKβ pathway to enable viral transcription activation and disable host antiviral cytokine gene expression echo findings from the current study.

KSHV RTA was originally reported to induce the degradation of IRF3 and IRF7 via the ubiquitin/proteasome-mediated pathway. Recent studies identified additional cellular transcriptional regulators that are targeted by KSHV RTA and γHV68 RTA for degradation, including RelA (39) and Hey1 (15, 38). Although it is clear that RTA expression is sufficient to induce the ubiquitination and degradation of these transcriptional regulators, it remains an open question whether RTA possesses intrinsic E3 ligase activity toward the substrates identified thus far. It is conceivable that the expanding list of cellular targets of RTA will be diversified with our increasing understanding of the role of RTA in viral infection. Among gammaherpesvirus RTA homologs, KSHV RTA, together with additional E1 and E2, suffices to assemble polyubiquitin chains on IRF7 in vitro, implying that RTA serves as a bona fide E3 ligase to accelerate protein turnover. Due to the challenge in purifying γHV68 RTA from bacterial and mammalian cells (infected or transfected) (our unpublished data), we were unable to determine whether γHV68 RTA has intrinsic E3 ligase activity toward RelA. In support of the conclusion that RTA may be a bona fide E3 ligase, a cysteine-rich domain is highly conserved within multiple RTA proteins of gammaherpesviruses, and mutations of two key cysteine residues greatly impaired RTA-induced RelA degradation. It is important that the RTA C/S variant demonstrated residual activity to induce RelA degradation and reduced RelA-dependent NF-κB activation. Furthermore, the RTA C/S variant was ubiquitinated more than wild-type RTA, particularly when cells were treated with MG132. This result indicates that the C/S mutations are not necessary for RTA ubiquitination, although they are essential for RTA-induced RelA ubiquitination and degradation. We surmise that the cysteine-rich domain may transfer a polyubiquitin chain from RTA to RelA, thereby promoting RelA degradation. As such, the loss of the ability to transfer polyubiquitin results in a greater ubiquitination of RTA itself. These results strongly argue that RTA recruits a cellular E3 ligase(s) to facilitate RelA degradation. Recently, KSHV RTA was shown to engage the cellular RAUL E3 ligase, promoting the ubiquitination and degradation of IRF7 (40). These observations bolster the conclusion that the induction of protein degradation is a key function of RTA, because multiple mechanisms exist to warrant the rapid turnover of RTA-targeted proteins.

In addition to viral factors that target RelA for degradation, recent studies have identified several cellular ubiquitin E3 ligases that are important for this process, including SOCS1 (29, 34), COMMD1 (14, 21, 22, 36), and PDLIM2 (35). Among them, SOCS1 and COMMD1, together with cullin and elongins B and C, assemble into a RING finger-containing ubiquitin E3 ligase complex. PDLIM2, a nuclear ubiquitin E3 ligase bearing PDZ and LIM domains, targets promoter-associated, transcriptionally active RelA and relocalizes RelA into the promyelocytic leukemia (PML) body for degradation by the proteasome in macrophages (35). The residual activity of the RTA C/S variant to promote RelA degradation implies the existence of an alternative mechanism by which RTA may employ a cellular E3 ligase(s) to promote the ubiquitination and degradation of RelA. Further experiments are necessary to investigate the potential cellular E3 ligase complex in RTA-induced RelA degradation and NF-κB termination.

ACKNOWLEDGMENTS

We thank L. Evan Reddick for critical reading of the manuscript.

This work is supported by the University of Texas Southwestern Endowed Scholar Program and grants from the National Institutes of Health (R01 CA134241 and DE021445). P. Feng is also supported by a research scholar grant from the American Cancer Society (RSG-11-162-01-MPC). We declare no competing interests.

Footnotes

Published ahead of print 30 November 2011

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