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. 2018 Nov 27;92(24):e01180-18.
doi: 10.1128/JVI.01180-18. Print 2018 Dec 15.

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

Christine Zimmermann  1 , Nicole Büscher  1 , Steffi Krauter  1 , Nadine Krämer  1 , Uwe Wolfrum  2 , Elisabeth Sehn  2 , Stefan Tenzer  3   4 , Bodo Plachter  5   4
Affiliations

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

Christine Zimmermann et al. J Virol. .

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.

Keywords: ISG15; ISGylation; cytomegalovirus; dense bodies; pUL25; pUL26; pp65; tegument.

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Figures

FIG 1
FIG 1
pUL25 is packaged into virions of different HCMV strains in a pp65-dependent manner. Label-free quantitative mass spectrometry was used to determine the amount of pUL25 in virions of different pp65-positive and pp65-negative HCMV strains. TB40-UL83 Stop is a derivative of strain TB40 with stop codons interrupting the pp65 open reading frame. KB14 is a derivative of the BADwt strain with a deletion of the pp65 open reading frame. Strains TB40 and TB40-UL83 Stop were evaluated using two independent biological replicates (preparation 1 [Prep 1] and preparation 2 [Prep 2]). The means for four technical replicates of each sample were measured in parts per million (ppm). Bars represent the standard error. Differences between samples were evaluated by Student’s t test. **, P < 0.001; *, P < 0.01. The results confirm and extend the results of a previous study from our laboratories (8).
FIG 2
FIG 2
Generation of a UL25 deletion mutant. (A) Schematic representation of the mutagenesis strategy used. The location of the UL25 gene with respect to neighboring genes is shown by arrows. Towne-ΔUL25 was generated by inserting a galK expression cassette into the UL25 open reading frame. The first 287 bp of UL25 remained in the BAC sequence in order to preserve the promoter region of the neighboring UL24 gene. (B) Quantitative PCR analysis of the genome replication of Towne-ΔUL25 versus that of Towne. Cells were infected in a way to achieve comparable genome copy numbers in the cells at 4 h after infection (reference point). Samples were drawn at the indicated time points and were subjected to quantitative TaqMan PCR analysis. The values represent the means for 3 technical replicates. Error bars are indicated. The data represent those from one of two biological replicates.
FIG 3
FIG 3
Analysis of Towne-ΔUL25 with respect to virion morphogenesis and UL24 transcription. (A) Purification of DBs, virions, and noninfectious enveloped particles (NIEPs) by glycerol-tartrate ultracentrifugation. The different fractions are indicated. (B) Separation of purified virion and DB fractions from the indicated strains by polyacrylamide gel electrophoresis (PAGE). Proteins were visualized by silver staining. Molecular weight markers and the putative position of pUL25 are indicated. (C to F) Mass spectrometry of the outer tegument protein composition of the virions and DBs of two different clones of Towne-ΔUL25 and of Towne. The means for three technical replicates of each sample were measured in parts per million (ppm), and the values are indicated on the y axis. Bars represent the standard error. (C and D) Proteome of virions; (E and F) proteome of DBs. Note the different scales in panels C and E versus panels D and F. A separate analysis confirming the lack of an impact of pUL25 expression on the overall protein composition of virions and DBs was performed (not shown). (G) Northern blot analysis of the transcription from the UL25-flanking gene UL24. Total cell RNA, prepared from 5-day-infected or mock-infected cells, was subjected to formaldehyde agarose gel electrophoresis. Following transfer, filters were hybridized to a UL24-specific probe. An RNA of 3.8 kb corresponding to a transcript terminating at the 3′ end of UL21a could be detected.
FIG 4
FIG 4
Transmission electron micrographs of cells infected with either Towne-ΔUL25 or Towne. (A and C) Images of cytoplasmic virion and DB formation in human foreskin fibroblasts infected with Towne-ΔUL25. (B and D) Images of cytoplasmic virion and DB formation in human foreskin fibroblasts infected with Towne. Bars indicate the diameters of the virions of both strains.
FIG 5
FIG 5
Immunoblot analysis of steady-state levels of pUL26 in infected cells. HFF cells were infected with Towne-ΔUL25 or Towne (A, B) or with Δpp65 or Towne (C, D) at an MOI of 1 (IE1 staining). After 6 days, cells were collected, lysed, and submitted to SDS-PAGE. (A, C) After transfer to PVDF membranes, immunoblot analysis was performed using antibodies against pUL26 and alpha-tubulin. (B, D) The levels of UL26 were quantified by measuring signal intensities with an ECL scanner, using Lab and Fiji ImageJ software for analysis. Tubulin was used as an internal reference. The images show one representative result out of three biological replicates.
FIG 6
FIG 6
Immunoblot analysis of pUL26 degradation in the absence of pUL25 or pp65. (A) HFF cells were infected with Towne-ΔUL25 or Towne-UL25-FLAG at an MOI of 1 (IE1 staining). (C) HFF cells were infected with Towne-Δpp65 or with Towne. After 6 days, some samples were treated with MG132 for 16 h, and whole-cell lysates were subsequently collected. SDS-PAGE and Western blot analyses were performed. Membranes were probed with pUL26- or FLAG tag-specific antibodies (A) or with pUL26- or pp65-specific antibodies (C). (B, D) The protein levels of pUL26 were quantified by measuring the signal intensities with an ECL scanner, using Lab and Fiji ImageJ software for analysis. Tubulin was used as an internal reference. Pictures show one representative result out of two biological replicates.
FIG 7
FIG 7
Analysis of pUL25 interaction with pUL26. (A) Coimmunoprecipitation of FLAG-tagged pUL25 of two independent biological replicates (samples 1 and 2). HFF were infected with Towne-UL25-FLAG at an MOI of 1 (IE1 staining) and were harvested after 6 days. Cell lysates were incubated with anti-FLAG-conjugated magnetic beads, and the precipitates were analyzed in a Western blot, probed with anti-FLAG and anti-UL26 antibodies. Uninfected HFF served as a negative control (Mock). (B) Coimmunoprecipitation of FLAG-tagged pUL25 of purified viral particles. Virions and DBs were purified as described in Materials and Methods, lysed, and analyzed in a Western blot. Lanes 1 and 5 refer to the amount of UL25-FLAG and UL26 present in the obtained lysates (Input). UL25-FLAG was then precipitated using anti-FLAG-conjugated magnetic beads. Precipitates were analyzed by use of a Western blot, probed with anti-FLAG and anti-UL26 antibodies. Lanes 2 and 6 display the coprecipitates of UL25-FLAG and UL26 after incubation with anti-FLAG magnetic beads and elution with 1 M glycine, pH 2.5. The supernatant (SN) shows the amounts of UL25-FLAG and UL26 that were not bound by anti-FLAG magnetic beads (lanes 3 and 5) as well as precipitates that were not eluted by glycine but that were eluted using Laemmli buffer (lanes 4 and 8).
FIG 8
FIG 8
Interferon-stimulated gene 15 protein (ISG15) expression and ISGylation in infected cells. (A) HFF cells were infected with Towne-ΔUL25 or Towne-UL25-FLAG at an MOI of 1 (IE1 staining). After 6 days, some samples were treated with 10 µM MG132 for 16 h and whole-cell lysates were subsequently collected. SDS-PAGE was performed, and the Western blot was probed against an ISG15 antibody. (B) HFF cells were infected with Towne-Δpp65 or Towne and were treated and processed as outlined in the legend to panel A. Free ISG15 and ISGylated proteins are indicated. The prominent band below the full-length ISG15 band likely represents an intermediate during proteasomal degradation that is stabilized by MG132 treatment. The results are those of one representative experiment out of two biological replicates.
FIG 9
FIG 9
Release of viral genomes from HFF infected with Towne or Towne-ΔUL25 in the presence or absence of IFN-β. HFF were infected with the different strains in a way in which roughly equal genome copy numbers were present at the beginning of the infection (measured by qPCR at 4 h postinfection). Cell culture supernatants were collected at the indicated times. The amount of viral genomes in these samples was measured by qPCR. The values in each sample are indicated in the figure, together with the relative values of reduction in cultures treated with IFN-β. All values are means for three technical replicates. The results shown represent those from one of two biological replicates.
FIG 10
FIG 10
ISG15 expression and ISGylation during HCMV infection. (a) HCMV infection leads to the initial induction of ISG15 expression. (b) This results in an increased conjugation of cellular and viral proteins with ISG15. (c) ISG15 expression and possibly ISGylation interfere with viral reproduction by insufficiently understood mechanisms. (d) The viral IE1 protein interferes with ISG15 induction and with ISGylation. (e) In addition, pUL50 interacts with UBE1L, an E1-activating enzyme for ISGylation, leading to its degradation and thus reducing protein ISGylation. (f, g), Deletion of either UL25 or UL83 (pp65) from the viral genome results in enhanced levels of ISG15 and ISGylation in infected cells. (h) pUL26 abrogates ISG15 expression and ISGylation at later stages of HCMV infection. (i) pUL25 interacts with pUL26 in the cell and in viral particles and prevents proteasomal degradation of pUL26. (j) Deletion of UL26 leads to an attenuation of virus release. (k) The lack of both pUL25 or pUL26 results in the enhanced susceptibility of HCMV infection to IFN-β.
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