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

Abstract

The tegument of human cytomegalovirus (HCMV) virions contains proteins that interfere with both the intrinsic and the innate immunity. One protein with a thus far unknown function is pUL25. The deletion of pUL25 in a viral mutant (Towne-ΔUL25) had no impact on the release of virions and subviral dense bodies or on virion morphogenesis. Proteomic analyses showed few alterations in the overall protein composition of extracellular particles. A surprising result, however, was the almost complete absence of pUL26 in virions and dense bodies of Towne-ΔUL25 and a reduction of the large isoform pUL26-p27 in mutant virus-infected cells. pUL26 had been shown to inhibit protein conjugation with the interferon-stimulated gene 15 protein (ISG15), thereby supporting HCMV replication. To test for a functional relationship between pUL25 and pUL26, we addressed the steady-state levels of pUL26 and found them to be reduced in Towne-ΔUL25-infected cells. Coimmunoprecipitation experiments proved an interaction between pUL25 and pUL26. Surprisingly, the overall protein ISGylation was enhanced in Towne-ΔUL25-infected cells, thus mimicking the phenotype of a pUL26-deleted HCMV mutant. The functional relevance of this was confirmed by showing that the replication of Towne-ΔUL25 was more sensitive to beta interferon. The increase of protein ISGylation was also seen in cells infected with a mutant lacking the tegument protein pp65. Upon retesting, we found that pUL26 degradation was also increased when pp65 was unavailable. Our experiments show that both pUL25 and pp65 regulate pUL26 degradation and the pUL26-dependent reduction of ISGylation and add pUL25 as another HCMV tegument protein that interferes with the intrinsic immunity of the host cell.IMPORTANCE Human cytomegalovirus (HCMV) expresses a number of tegument proteins that interfere with the intrinsic and the innate defense mechanisms of the cell. Initial induction of the interferon-stimulated gene 15 protein (ISG15) and conjugation of proteins with ISG15 (ISGylation) by HCMV infection are subsequently attenuated by the expression of the viral IE1, pUL50, and pUL26 proteins. This study adds pUL25 as another factor that contributes to suppression of ISGylation. The tegument protein interacts with pUL26 and prevents its degradation by the proteasome. By doing this, it supports its restrictive influence on ISGylation. In addition, a lack of pUL25 enhances the levels of free ISG15, indicating that the tegument protein may interfere with the interferon response on levels other than interacting with pUL26. Knowledge obtained in this study widens our understanding of HCMV immune evasion and may also provide a new avenue for the use of pUL25-negative strains for vaccine production.

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.…”

Constitutive immune mechanisms: mediators of host defence and immune regulation

“…infections 109 , and the ubiquitin–proteasome pathway is targeted by many viruses and bacteria to promote replication 110 – 114 . For example, the human cytomegalovirus protein pUL25 inhibits proteasomal degradation of another viral protein, pUL26, to sustain the activity of a pUL26-mediated immune evasion mechanism 114 . Collectively, these examples show that the conserved proteasome pathway is part of the constitutive immune defence repertoire.…”

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).…”