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the viral load of recombinant γHV68.C/S at 4 and 7 days p.i. was under the detection limit by a plaque assay (our unpublished data).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
). 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
), 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
). 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
), COMMD1 (14
), 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.