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. IMPORTANCE 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.

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The Human Cytomegalovirus Transmembrane Protein pUL50 Induces Loss of VCP/p97 and Is Regulated by a Small Isoform of pUL50

Journal of Virology ◽

10.1128/jvi.00110-20 ◽

2020 ◽

Vol 94 (13) ◽

Author(s):

Myoung Kyu Lee ◽

Seokhwan Hyeon ◽

Jin-Hyun Ahn

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Amino Acid Position ◽

Mutant Virus ◽

Early Gene ◽

Immediate Early ◽

Nuclear Egress ◽

The Unfolded Protein Response

ABSTRACT The human cytomegalovirus (HCMV) UL50 gene encodes a transmembrane protein, pUL50, which acts as a core component of the nuclear egress complex (NEC) for nucleocapsids. Recently, pUL50 has been shown to have NEC-independent activities: downregulation of IRE1 to repress the unfolded protein response and degradation of UBE1L to inhibit the protein ISG15 modification pathway. Here, we demonstrate that a 26-kDa N-terminal truncated isoform of pUL50 (UL50-p26) is expressed from an internal methionine at amino acid position 199 and regulates the activity of pUL50 to induce the loss of valosin-containing protein (VCP/p97). A UL50(M199V) mutant virus expressing pUL50(M199V) but not UL50-p26 showed delayed growth at a low multiplicity of infection. There was also delayed accumulation of the viral immediate early 2 (IE2) protein in the mutant virus, and this correlated with the reduced expression of VCP/p97, which promotes IE2 expression. Infection with mutant virus did not significantly alter ISGylation levels. In transient expression assays, pUL50 induced VCP/p97 loss posttranscriptionally, and this was dependent on the presence of its transmembrane domain. In contrast, UL50-p26 did not destabilize VCP/p97 but, rather, inhibited pUL50-mediated VCP/p97 loss and the associated major IE gene suppression. Both pUL50 and UL50-p26 interacted with VCP/p97, although UL50-p26 did so more weakly than pUL50. UL50-p26 interacted with pUL50, and this interaction was much stronger than the pUL50 self-interaction. Furthermore, UL50-p26 was able to interfere with the pUL50-VCP/p97 interaction. Our study newly identifies UL50-p26 expression during HCMV infection and suggests a regulatory role for UL50-p26 in blocking pUL50-mediated VCP/p97 loss by associating with pUL50. IMPORTANCE Targeting the endoplasmic reticulum (ER) by viral proteins may affect ER-associated protein homeostasis. During human cytomegalovirus (HCMV) infection, pUL50 targets the ER through its transmembrane domain and moves to the inner nuclear membrane (INM) to form the nuclear egress complex (NEC), which facilitates capsid transport from the nucleus to the cytoplasm. Here, we demonstrate that pUL50 induces the loss of valosin-containing protein (VCP/p97), which promotes the expression of viral major immediate early gene products, in a manner dependent on its membrane targeting but that a small isoform of pUL50 is expressed to negatively regulate this pUL50 activity. This study reports a new NEC-independent function of pUL50 and highlights the fine regulation of pUL50 activity by a smaller isoform for efficient viral growth.

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ISG12a Restricts Hepatitis C Virus Infection through the Ubiquitination-Dependent Degradation Pathway

Journal of Virology ◽

10.1128/jvi.00352-16 ◽

2016 ◽

Vol 90 (15) ◽

pp. 6832-6845 ◽

Cited By ~ 18

Author(s):

Binbin Xue ◽

Darong Yang ◽

Jingjing Wang ◽

Yan Xu ◽

Xiaohong Wang ◽

...

Keyword(s):

Innate Immunity ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Infection ◽

Viral Protein ◽

E3 Ligase ◽

Antiviral Effect ◽

Degradation Pathway ◽

Viral Spread ◽

Ubiquitin E3 Ligase

ABSTRACTInterferons (IFNs) restrict various kinds of viral infection via induction of hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high-IFN-inducible gene, ISG12a (also known as IFI27), exhibits a nonapoptotic antiviral effect on hepatitis C virus (HCV) infection. Viral NS5A protein is targeted specifically by ISG12a, which mediates NS5A degradation via a ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. S-phase kinase-associated protein 2 (SKP2) is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity.IMPORTANCEUpon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadly unclear. The present study shows a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase, SKP2, for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity.

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Hls5, a Novel Ubiquitin E3 Ligase, Modulates Levels of Sumoylated GATA-1.

Blood ◽

10.1182/blood.v114.22.253.253 ◽

2009 ◽

Vol 114 (22) ◽

pp. 253-253 ◽

Cited By ~ 1

Author(s):

Louise Natalie Winteringham ◽

Raelene Endersby ◽

Jennifer Beaumont ◽

Jean-Philippe Lalonde ◽

Merlin Crossley ◽

...

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Coiled Coil ◽

Ring Finger ◽

E3 Ligase ◽

Proteasomal Degradation ◽

Post Translational Modifications ◽

E3 Ligases ◽

Lineage Switch ◽

Ubiquitin E3 Ligase

Abstract Abstract 253 Hemopoietic lineage commitment is controlled, in part, by transcription factors that regulate specific genes required for the formation of mature blood cells. Differentiation along particular hemopoietic lineages is dependant not only on the presence of particular transcription factors, but also on appropriate concentrations - altering transcription factor levels can force cells into different hemopoietic pathways. Transcription factors undergo numerous post-translational modifications and are controlled spatially via sub-cellular localisation. De-regulation of transcription factors can result in leukemias, or other blood disorders. GATA-1 is an example of a key lineage-determining gene, essential for erythropoiesis. Increasing GATA-1 levels promotes maturation along the erythroid pathway, whereas reducing GATA-1 concentrations favours myelopoiesis. GATA-1 regulation occurs at multiple levels including transcription, translation and post-translational modifications such as phosphorylation, acetylation, ubiquitination and sumoylation. Although GATA-1 ubiquitination modifies the protein for proteasomal degradation, the effect of adding small ubiquitin-like modier (Sumo) to GATA-1 is unclear. Several examples of hemopoietic differentiation plasticity have been observed. We reported a lineage switch by erythroleukemic J2E cells which spontaneously developed a monoblastoid phenotype. Two genes (Hls5 and Hls7/Mlf1) were isolated from this lineage switch with potential lineage-determining features. Hls5 is a member of the RBCC (Ring finger, B-box, Coiled-coil) family of proteins, which includes PML. Ectopic expression of Hls5 impedes erythroid differentiation by reducing GATA-1 levels, and suppressing hemoglobin synthesis. Significantly, Hls5 relocates from the cytoplasm to associate with GATA-1 in the nucleus, where it interferes with DNA binding and transactivation of GATA-1. Several members of the RBCC family are ubiquitin E3 ligases, catalysing the final step in the ubiquitination process - these molecules play a vital role in regulating the levels of target proteins. Here we show that Hls5 is a bona fide ubiquitin E3 ligase, in partnership with several ubiquitin E2 enzymes. The Ring finger is critical for Hls5 ligase activity as mutation of key residues within the Ring finger ablates catalytic activity. Interestingly, a yeast 2 hybrid screen for Hls5 interactors identified Ubc9 and Pias1, which act as E2 and E3 enzymes in the sumoylation cascade. Co-immunoprecipitation, BRET and co-localization experiments confirmed the Hls5 association with Ubc9 and Pias1. Moreover, Hls5 binds Sumo-1 (but not Sumo-2 or 3), and co-localizes with Sumo-1 in discrete nuclear bodies. Thus, Hls5 interacts with several components of the intracellular sumoylation machinery. Hls5 can also reduce sumoylated proteins globally, indicating it may target these modified proteins for degradation. Recently, a new family of ubiquitin E3 ligases has been described which specifically mark sumoylated proteins for degradation. These Sumo-targeted ubiquitin ligases (STUbL) are found primarily in yeast, and only one mammalian STUbL has been identified. We postulated that Hls5 may be a STUbL, capable of regulating sumoylated GATA-1. Our data demonstrate that while Hls5 is able to bind GATA-1 via the B-box and Coiled-coil domains, it preferentially associates with sumoylated GATA-1 through a canonical Sumo interacting motif (SIM). This results in increased GATA-1 ubiquitination and, as a consequence, levels of sumoylated GATA-1 are reduced substantially. Since mutation of the lysine necessary for Sumo attachment does not affect GATA-1 transactivation, sumoylation may act as a prelude to ubiquitination and protein turn-over. We propose, therefore, that GATA-1 mediates transcription of target genes, and is subsequently sumoylated by Pias1 and Ubc9 – addition of Sumo moieties to GATA-1 enhance binding to Hls5, which in turn impedes GATA-1 DNA binding, and promotes ubiquitination for proteasomal degradation. This model is consistent with decreased levels of GATA-1 in erythroid cells ectopically expressing Hls5, and with the original isolation of Hls5 as a potential lineage-determining gene involved with the erythroid to monoblastoid lineage switch. Thus, Hls5 is a novel STUbL which plays a role in hemopoietic lineage commitment by modulating GATA-1 activity and content. Disclosures: No relevant conflicts of interest to declare.

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The ubiquitin E3 ligase CHIP promotes proteasomal degradation of the serine/threonine protein kinase PINK1 during staurosporine-induced cell death

Journal of Biological Chemistry ◽

10.1074/jbc.m117.803890 ◽

2017 ◽

Vol 293 (4) ◽

pp. 1286-1297 ◽

Cited By ~ 7

Author(s):

Lang Yoo ◽

Kwang Chul Chung

Keyword(s):

Cell Death ◽

Protein Kinase ◽

E3 Ligase ◽

Proteasomal Degradation ◽

Ubiquitin E3 Ligase

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Degradation of host ubiquitin E3 ligase Itch by human cytomegalovirus UL42

Journal of General Virology ◽

10.1099/jgv.0.000336 ◽

2016 ◽

Vol 97 (1) ◽

pp. 196-208 ◽

Cited By ~ 10

Author(s):

Tetsuo Koshizuka ◽

Keiichiro Tanaka ◽

Tatsuo Suzutani

Keyword(s):

Human Cytomegalovirus ◽

E3 Ligase ◽

Ubiquitin E3 Ligase

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MITOL-mediated DRP1 ubiquitylation and degradation promotes mitochondrial hyperfusion in CMT2A-linked MFN2 mutant

Journal of Cell Science ◽

10.1242/jcs.257808 ◽

2021 ◽

Author(s):

Rajdeep Das ◽

Izaz Monir Kamal ◽

Subhrangshu Das ◽

Saikat Chakrabarti ◽

Oishee Chakrabarti

Keyword(s):

E3 Ligase ◽

Proteasomal Degradation ◽

Mitochondrial Morphology ◽

Charcot Marie Tooth ◽

Differential Interaction ◽

Ubiquitin E3 Ligase ◽

Mechanistic Insight ◽

Tooth Type ◽

Insight Into

Mutations in Mitofusin2 (MFN2), associated with the pathology of the debilitating neuropathy, Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. One such abundant MFN2 mutant, R364W results in the generation of elongated, interconnected mitochondria. However, the mechanism leading to this mitochondrial aberration remains poorly understood. Here we show that mitochondrial hyperfusion in the presence of R364W-MFN2 is due to increased degradation of DRP1. The Ubiquitin E3 ligase MITOL is known to ubiquitylate both MFN2 and DRP1. Interaction with and its subsequent ubiquitylation by MITOL is stronger in presence of WT-MFN2 than R364W-MFN2. This differential interaction of MITOL with MFN2 in the presence of R364W-MFN2 renders the ligase more available for DRP1 ubiquitylation. Multimonoubiquitylation and proteasomal degradation of DRP1 in R364W-MFN2 cells in the presence of MITOL eventually leads to mitochondrial hyperfusion. Here we provide a mechanistic insight into mitochondrial hyperfusion, while also reporting that MFN2 can indirectly modulate DRP1 – an effect not shown before.

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Irf2bp2a regulates terminal granulopoiesis through proteasomal degradation of Gfi1aa in zebrafish

PLoS Genetics ◽

10.1371/journal.pgen.1009693 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009693

Author(s):

Shuo Gao ◽

Zixuan Wang ◽

Luxiang Wang ◽

Haihong Wang ◽

Hao Yuan ◽

...

Keyword(s):

Drug Targets ◽

Ring Finger ◽

E3 Ligase ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Link Type ◽

Ubiquitin E3 Ligase ◽

Anti Cancer ◽

Ubiquitin Proteasome ◽

Transcription Regulators

The ubiquitin-proteasome system plays important roles in various biological processes as it degrades the majority of cellular proteins. Adequate proteasomal degradation of crucial transcription regulators ensures the proper development of neutrophils. The ubiquitin E3 ligase of Growth factor independent 1 (GFI1), a key transcription repressor governing terminal granulopoiesis, remains obscure. Here we report that the deficiency of the ring finger protein Interferon regulatory factor 2 binding protein 2a (Irf2bp2a) leads to an impairment of neutrophils differentiation in zebrafish. Mechanistically, Irf2bp2a functions as a ubiquitin E3 ligase targeting Gfi1aa for proteasomal degradation. Moreover, irf2bp2a gene is repressed by Gfi1aa, thus forming a negative feedback loop between Irf2bp2a and Gfi1aa during neutrophils maturation. Different levels of GFI1 may turn it into a tumor suppressor or an oncogene in malignant myelopoiesis. Therefore, discovery of certain drug targets GFI1 for proteasomal degradation by IRF2BP2 might be an effective anti-cancer strategy.

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