Consecutive Inhibition of ISG15 Expression and ISGylation by Cytomegalovirus Regulators

Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNβ treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection.
Author Summary Type I IFN response is a front-line defense against virus infection. Activation of type I IFN signaling leads to expression of a subset of cellular proteins encoded by interferon-stimulated genes (ISGs). ISG15 encodes an ubiquitin-like protein that is covalently conjugated to protein lysine residues. ISG15 modification (ISGylation) of a protein causes changes of protein function. ISGylation is known to inhibit the replication of many viruses, although pro-viral effects of ISGylation are also reported. Given that ISG15 and the enzymes involved in ISGylation are strongly induced upon virus infection, understanding the interplay between virus and ISGylation is an important issue in virus-host interaction. Nevertheless, viral substrates of ISG15 and viral strategies to regulate ISGylation-mediated antiviral responses are limited to only a few examples. In this study we demonstrate that ISGylation suppresses human cytomegalovirus (HCMV) infection but the virus is armed with countermeasures that consecutively reduce ISG15 transcription and protein ISGylation. Interestingly, a viral ISG15 target is found to inhibit ISGylation. This study highlights that ISGylation is a critical innate immune response against HCMV infection and interfering with ISG15-mediated anti-viral immunity is critical for productive viral infection.