Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L

Abstract

Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and (to a lesser extent) with ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon beta treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L in a manner independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an endoplasmic reticulum (ER)-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation. Proteins can be conjugated covalently by ubiquitin or ubiquitin-like proteins, such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses, including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of UBE1L, an E1-activating enzyme for ISGylation. This pUL50 activity requires membrane targeting. In support of this finding, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence, to our knowledge, that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.

Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression

“…This finding is an essential addition to the study of ISG15 in the context of not only HCMV infection but other viruses as well. It adds complexity to recent work describing ISG15 as an antiviral effector during HCMV infection that is antagonized by IE1, pUL50, pUL26 and pUL25 [27,52,53,54], and potentially explains the persistence of ISG15 even in cells treated with high concentrations of the JAK inhibitor pyridone-6 [27].…”

“…In a broader sense, the results of our study raise questions about the role of IFN-independent ISGs in host responses to HCMV. ISG15 appears to be primarily antiviral [27,52,53,54] however, viperin is known to enhance viral replication by allowing HCMV to regulate cellular lipid metabolism [32,57]. This is not the case for other viruses as viperin mainly functions as an antiviral ISG [58,59], and demonstrates the potential for investigations into IFN-independent ISGs to illustrate new functions of well-known host defense proteins.…”

Interferon-Independent Innate Responses to Cytomegalovirus

“…During HCMV infection accumulation of both free and conjugated ISG15 can be partially inhibited by interfering with the canonical IFNAR signaling pathway with a JAK inhibitor (115) but some IFN-independent, IRF3-dependent expression remains (96). Whilst the mechanisms by which ISG15 regulates CMV infection are currently unknown, it appears to possess antiviral activity as blocking ISG15 accumulation enhances viral replication (115) and HCMV antagonizes both the production of unconjugated ISG15 and ISGylation (115)(116)(117)(118).…”

The Abundant Tegument Protein pUL25 of Human Cytomegalovirus Prevents Proteasomal Degradation of pUL26 and Supports Its Suppression of ISGylation

“…For HCMV, IE1 and, more recently, pUL50 have been described, besides pUL26, to interfere with these processes (Fig. 10) (16,27,37). IE1 was found to reduce ISG15 expression by impairing cytosolic DNA sensing by cGAS/STING (27).…”

“…IE1 was found to reduce ISG15 expression by impairing cytosolic DNA sensing by cGAS/STING (27). pUL50 was shown to interact with UBE1L, an E1-activating enzyme for ISGylation, leading to its degradation (37). pUL26 was initially identified to be a transcriptional transactivator (15).…”