Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 1 Interacts with a Member of the Interferon-Stimulated Gene 15 Pathway

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus known to establish lifelong latency in the human host. We and others have previously shown that three KSHV homologs of cellular interferon regulatory factors (IRFs), known as viral IRFs (vIRFs), participate in evasion of the host interferon (IFN) response. We report that vIRF1 interacts with the cellular interferon-stimulated gene 15 (ISG15) E3 ligase, HERC5, in the context of Toll-like receptor 3 (TLR3) activation and IFN induction. The ISG15 protein is covalently conjugated to target proteins upon activation of the interferon response. Interaction between vIRF1 and HERC5 was confirmed by immunoprecipitation, and the region between amino acids 224 and 349 of vIRF1 was required for interaction with HERC5. We further report that expression of vIRF1 in the context of TLR3 activation results in decreased ISG15 conjugation of proteins. Specifically, TLR3-induced ISG15 conjugation and protein levels of cellular IRF3, a known ISG15 target, were decreased in the presence of vIRF1 compared to the control. vIRF1 itself was also identified as a target of ISG15 conjugation. KSHV-infected cells exhibited increased ISG15 conjugation upon reactivation from latency in coordination with increased IFN. Furthermore, knockdown of ISG15 in latently infected cells resulted in a higher level of KSHV reactivation and an increase in infectious virus. These data suggest that the KSHV vIRF1 protein affects ISG15 conjugation and interferon responses and may contribute to effective KSHV replication. IMPORTANCEThe KSHV vIRF1 protein can inhibit interferon activation in response to viral infection. We identified a cellular protein named HERC5, which is the major ligase for ISG15, as a vIRF1 binding partner. vIRF1 association with HERC5 altered ISG15 modification of cellular proteins, and knockdown of ISG15 augmented reactivation of KSHV from latency. U pon detection of viral infection, cells activate the quintessential antiviral immune response, which results in production of type I interferon (IFN), including interferon alpha (IFN-␣) and IFN-␤. Type I IFN induces an antiviral transcriptional program, producing proteins that cooperate to inhibit the spread of infection. One of the most abundantly produced transcripts upon induction of the type I IFN response is interferon-stimulated gene 15 (ISG15) (1, 2). ISG15 is a ubiquitin-like 17-kDa protein that is covalently conjugated to target proteins via a process called ISGylation (3-5). Similar to ubiquitin, ISG15 requires three enzymatic steps for conjugation onto target proteins. They include the E1 enzyme Ube1L, the conjugating E2 enzyme UbcH8, and the major E3 ligase HERC5 (6). Multiple large-scale screens have identified hundreds of potential cellular targets of ISGylation; however, in most cases, the specific function of ISG15 conjugation to cellular proteins remains undetermined (7).Thus far, identified ISG15 target proteins function in a variety of cellular pathways, including glycolysis, cell motility, translat...

ISG15 in antiviral immunity and beyond

“…One proposed mechanism for this was through the regulation of specific KSHV microRNAs that are known to modulate KSHV latency 74 . In a second study, KSHV infection was reactivated in primary effusion lymphoma cells by various means, and both ISG15 and ISG15 conjugate levels were found to be increased, along with activation of the type I interferon system 75 . Knockdown of either ISG15 or the E3 ligase HERC5 resulted in an increase in KSHV reactivation and an increase in the production of infectious virus 75 .…”

Transcriptomic analysis of KSHV-infected primary oral fibroblasts: The role of interferon-induced genes in the latency of oncogenic virus

“…We found that silencing of either ISG15 or ISG20 greatly increased the virion release (there were more Lana transcripts in these infected groups compared to controls) (Figure 3C). Interestingly, one very recent study also reported that silencing of ISG15 in KSHV latently infected iSLK.219 cells resulted in a higher level of virus reactivation and an increase in infectious virus production [20]. They also found that KSHV-encoded vIRF1 protein can inhibit IFN activation in response to viral infection, through interaction with HERC5, an ISG15 E3 ligase, to alter ISG15 modification of cellular proteins [20].…”

“…Interestingly, vIRF1 itself was also a target of ISG15 conjugation. KSHV-infected cells exhibited increased ISG15 conjugation upon reactivation from latency in coordination with increased IFN [20]. …”

Viral Inhibition of PRR-Mediated Innate Immune Response: Learning from KSHV Evasion Strategies

“…Interestingly, among vIRFs, only vIRF1 decreases phosphorylation and nuclear translocation of IRF3 upon TLR3 activation, suggesting that vIRF1 and other vIRFs block TLR3-mediated IFN-β production in different manners (Jacobs et al, 2013). Recently, Jacobs et al (2015) demonstrated that vIRF1 interacts with cellular ISG15 E3 ligase HERC5 and decreases global IS-Glyation in the context of TLR3 activation. Together, these findings suggest that each vIRF may uniquely inhibit the TLR3-mediated antiviral responses.…”