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. 2018 Jul 17;92(15):e00462-18.
doi: 10.1128/JVI.00462-18. Print 2018 Aug 1.

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L

Myoung Kyu Lee  1 , Ye Ji Kim  1 , Young-Eui Kim  1 , Tae-Hee Han  1 , Jens Milbradt  2 , Manfred Marschall  2 , Jin-Hyun Ahn  3
Affiliations

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L

Myoung Kyu Lee et al. J Virol. .

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.

Keywords: ISGylation; UBE1L; UL50; cytomegalovirus.

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Figures

FIG 1
FIG 1
Interaction of pUL50 with UBE1L and ISG15. (A) 293T cells in six-well plates were cotransfected with plasmids encoding pUL50-HA (0.5 μg) and expressing Myc-tagged UBE1L (1 μg), UbcH8 (0.5 μg), Herc5 (1 μg), or ISG15AA (1 μg), as indicated. Forty-eight hours after transfection, cell lysates were prepared and immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) with anti-HA antibody. The expression level of each protein in total cell lysate was also determined by immunoblotting. A small isoform of pUL50, of approximately 26 kDa (UL50-p26), was also detected. The amounts of pUL50-HA proteins coimmunoprecipitated relative to the input amounts of proteins were quantitated, and the relative binding strengths are shown in the graph. (B) HF cells in 150-mm dishes were infected with recombinant HCMV (AD169) containing the UL50-HA gene at a multiplicity of infection (MOI) of 3. Seventy-two hours after infection, cell lysates were prepared and immunoprecipitated with anti-HA or anti-Flag (as a negative control) antibody, followed by immunoblotting with anti-UBE1L. (C) Similar immunoprecipitation was performed 48 h after infection, followed by immunoblotting with anti-ISG15 antibody. The expression level of each protein in total cell lysate was also determined by immunoblotting. Levels of β-actin are shown as a loading control.
FIG 2
FIG 2
Effect of pUL50 expression on levels of ISGylation. 293T cells in six-well plates were cotransfected with plasmids (0.25 μg) expressing Myc-ISG15GG, HA-UBE1L, Flag-UbcH8, and HA-Herc5 and increasing amounts (0.2, 0.5, and 1 μg) of a plasmid expressing SRT-pUL50, as indicated. Forty-eight hours after transfection, cell lysates were prepared and subjected to immunoblotting with anti-Myc or anti-SRT antibody. Levels of β-actin are shown as a loading control.
FIG 3
FIG 3
Effect of pUL50 expression on levels of ISGylation enzymes. (A to E) 293T cells in six-well plates were cotransfected with a plasmid (1 μg) expressing HA-UBE1L (A and E), HA-Herc5 (B), Flag-UbcH8 (C), or Myc-ISG15AA (D) and increasing amounts (0.04, 0.2, and 1 μg) of a plasmid expressing pUL50-HA (A to D) or SRT-pUL26 (E), as indicated. Twenty-four hours after transfection, cells were left untreated or treated with MG132 (20 μM) for 24 h, and immunoblotting was performed as indicated. Levels of β-actin are shown as a loading control. The amounts of the UA-UBE1L, HA-Herc5, Flag-UbcH8, and Myc-ISG15AA proteins relative to those of β-actin are indicated below the blots. (F) 293T cells in six-well plates (two separate sets) were cotransfected with a plasmid expressing HA-UBE1L and increasing amounts of a plasmid expressing pUL50-HA, as for panel A. Forty-eight hours after transfection, cell lysates from one set were prepared for immunoblotting, while mRNAs from the other set were prepared for RT-PCR analysis. Immunoblotting was performed as described for panel A. The results of RT-PCR, showing the levels of UBE1L and β-actin mRNAs, are also shown.
FIG 4
FIG 4
Effect of pUL50 expression on UBE1L level induced by IFN-β treatment or viral infection. (A) HF cells were mock infected or infected with UV-HCMV or HCMV (Toledo strain) at an MOI of 3 for 24 or 48 h. The levels of the UBE1L, IE1/IE2, and pUL50 proteins were determined by immunoblotting. Bands indicating pUL50 and its 26-kDa isoform (UL50-p26) are indicated with arrowheads. Levels of β-actin are shown as a loading control. (B) Control and pUL50-HA-expressing HF cells produced by retroviral vectors (MIN) were left untreated or treated with IFN-β (1,000 U/ml) for 48 h and then incubated with or without cycloheximide (CHX; 100 μg/ml) for the indicated times. The levels of UBE1L, pUL50-HA, and β-actin were detected by immunoblotting. The relative levels of UBE1L (normalized to levels of β-actin) are shown in the graph. (C) HF cells were mock infected or infected with recombinant adenoviruses (Ad-lacZ or Ad-UL50-HA) at an MOI of 5 for 24 h and then superinfected with UV-HCMV (Toledo) for 24 h. The levels of UBE1L, pUL50-HA, and β-actin were determined by immunoblotting.
FIG 5
FIG 5
Requirement of the TM domain for ISGylation inhibition by pUL50. (A) 293T cells in six-well plates were cotransfected with plasmids (0.25 μg) expressing Myc-ISG15GG, HA-UBE1L, Flag-UbcH8, and HA-Herc5 and a plasmid (1 μg) expressing pUL50-HA (wild type [Wt] or the ΔTM or 1-358 mutant), as indicated. Forty-eight hours after transfection, immunoblotting was performed with anti-Myc or anti-HA antibody. Levels of β-actin are shown as a loading control. The positions of the wild-type pUL50 protein and its 26-kDa isoform (UL50-p26) are indicated. (B) Structures of the UL50 constructs used for panel A. The α-helix, globular domain, and transmembrane (TM) regions and the UL53 binding region are indicated. The relatively conserved regions (CR1 and CR2) in other herpesvirus homologs are also indicated. The numbers indicate amino acid positions. (C) 293T cells in six-well plates were cotransfected with plasmids encoding pUL50-HA (wild type or mutant) (1 μg) and Myc-UBE1L (0.5 μg), as indicated. Forty-eight hours after transfection, cell lysates were prepared and immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-Myc antibody. The expression level of each protein in total cell lysate was also determined by immunoblotting with anti-UBE1L or anti-HA antibody. The position of endogenous (endo.) UBE1L is indicated. Levels of β-actin are shown as a loading control. (D) HF cells in chamber slides were transfected with UL50 plasmids. Forty-eight hours after transfection, cells were fixed with 4% paraformaldehyde and stained with anti-HA antibody. Hoechst dye was used to stain cell nuclei. Representative confocal laser scanning microscopic images are shown.
FIG 6
FIG 6
Colocalization of UL50 proteins and UBE1L in MG132-treated cells. (A) HeLa cells in chamber slides were singly transfected with a plasmid containing the UL50-HA gene (wild type or ΔTM) or Myc-UBE1L. Forty-eight hours after transfection, double-label IFA was performed with anti-HA and anti-Myc antibodies. Hoechst dye was used to stain cell nuclei. (B) HeLa cells were cotransfected with plasmids expressing the Myc-UBE1L and UL50-HA proteins (Wt or ΔTM mutant), as indicated. Forty-eight hours after transfection, cells were left untreated or treated with MG132 (5 μM) for 16 h, and double-label IFA was performed as described for panel A. Representative confocal microscopic images are shown.
FIG 7
FIG 7
Involvement of RNF170 in pUL50-mediated UBE1L degradation. (A and B) 293T cells in six-well plates were cotransfected with plasmids expressing HA-UBE1L (0.5 μg) or pUL50-HA (0.25 μg) and a plasmid (2 μg) expressing RNF170v1-3×Flag (A) or RNF170v3-3×Flag or GFP-RNF170v3 (B), as indicated. Twenty-four hours after transfection, immunoblotting was performed as indicated. Levels of β-actin are shown as a loading control. The amounts of HA-UBE1L relative to those of β-actin are indicated under blots. (C) 293T cells were cotransfected with plasmids expressing pUL50-HA (0.5 μg) and RNF170v1-3×Flag or RNF170v3-3×Flag (2 μg), as indicated. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-HA antibody. The expression level of each protein in total cell lysate was also determined by immunoblotting. The amounts of pUL50-HA proteins coimmunoprecipitated relative to the input amounts of proteins were quantitated, and relative binding strengths are indicated under the blot. A diagram comparing the structures of RNF170v1 and RNF170v3 is shown. RNF170v1 and RNF170v3 share the N-terminal 133 amino acids. RNF170v1 contains two more transmembrane domains (orange boxes) in its C-terminal region.
FIG 8
FIG 8
Colocalization of RNF170 with UL50 proteins. (A) HeLa cells in chamber slides were singly transfected with plasmids expressing RNF170v1-3×Flag (a) or RNF170v3-3×Flag (b) or cotransfected with plasmids containing the UL50-HA gene and expressing RNF170v1-3×Flag (c) or RNF170v3-3×Flag (d). Forty-eight hours after transfection, double-label IFA was performed with anti-HA and anti-Flag antibodies. Hoechst dye was used to stain cell nuclei. Representative confocal microscopic images are shown. (B) HF cells in chamber slides were transfected via electroporation with a plasmid expressing RNF170v1-3×Flag. Twenty-four hours after transfection, cells were mock infected (a) or infected with UL50-HA virus at an MOI of 3 (b) for 120 h. Double-label IFA was performed as described for panel A.
FIG 9
FIG 9
Effects of UL50 and RNF170 on UBE1L ubiquitination. 293T cells in six-well plates were cotransfected with plasmids expressing Myc-UBE1L (0.25 μg), HA-Ub (0.5 μg), SRT-pUL50 (0.5 μg), and RNF170v1-3×Flag (1.5 μg), as indicated. Twenty-four hours after transfection, total cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-HA antibody to determine the ubiquitination level of UBE1L. The total protein levels of the Myc-UBE1L, SRT-pUL50, and RNF170v1-3×Flag proteins in cell lysates were also determined by immunoblotting with anti-Myc, anti-pUL50, and anti-Flag antibodies, respectively. The relative levels of ubiquitinated UBE1L are shown in the graph.
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