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. 2018 Apr 13;92(9):e00013-18.
doi: 10.1128/JVI.00013-18. Print 2018 May 1.

Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization

Gregor Maschkowitz  1   2 , Sabine Gärtner  2 , Heike Hofmann-Winkler  1   2 , Helmut Fickenscher  1 , Michael Winkler  3   2
Affiliations

Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization

Gregor Maschkowitz et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans-Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization.IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.

Keywords: UL35; cytomegalovirus; glycoprotein B; sorting nexin 5.

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Figures

FIG 1
FIG 1
Interaction of UL35 proteins with sorting nexin 5. (A) 293T cells were transfected with expression plasmids for ppUL35, ppUL35A, and FLAG-SNX5 or FLAG-SNX16, as indicated. After 48 h, cell extracts were precipitated with mouse monoclonal antibodies against FLAG (M2). Precipitated proteins were separated by polyacrylamide gel electrophoresis, and coprecipitated UL35 proteins were detected with a specific rabbit antiserum. Expression of the respective proteins in the cell lysates was controlled by immunoblotting (bottom). (B) Cell lines permissive for HCMV were tested for endogenous expression of SNX5. Cell lysates prepared from equal numbers of cells (4 × 104) were subjected to SDS-PAGE and immunoblotting. Endogenous SNX5 was detected by a polyclonal goat anti-SNX5 serum. (C) Coimmunoprecipitation of endogenous proteins from infected cells. HFF were mock treated or infected with TB4-wt at an MOI of 1 and harvested at 120 h postinfection. Cell lysates prepared from 6 × 106 cells were subjected to immunoprecipitation with goat anti-SNX5 serum. Precipitates and lysates were separated by SDS-PAGE and blotted, and proteins were detected with rabbit anti-UL35 serum as the primary antibody. (D) Kinetics of SNX5 expression during infection. HFF cells were mock treated or infected with TB4-wt at an MOI of 1, and cell lysates were prepared at the indicated time points (in hours postinfection). After SDS-PAGE and immunoblotting, proteins were detected with goat anti-SNX5 (top) or anti-β-actin primary antibodies.
FIG 2
FIG 2
Functional interaction of UL35 proteins with sorting nexin 5. (A) In a macropinocytosis assay, HeLa cells were transfected with an expression plasmid for FLAG-SNX5 and an expression plasmid for ppUL35 or ppUL35A. At 24 h after transfection, the cells were serum starved for 12 h. Cells were incubated for 3 min with EGF (to increase the basal macropinocytosis rate) and FITC-dextran and subsequently detached and fixed with paraformaldehyde. Uptake of FITC-dextran was measured by quantitation of positive cells by flow cytometry. Results are representative of those from at least 3 independent uptake assays; error bars indicate standard deviations. Significance was calculated employing a two-tailed Student's t test with the Welch correction. (B) To analyze the effects of UL35 proteins on the subcellular localization of CI-M6PR, HeLa cells were transfected with expression plasmids for EYFP-UL35 or EYFP-UL35A or for EYFP-UL82 as a control. At 24 h after transfection, cells were fixed with paraformaldehyde and stained with a specific antibody directed against CI-M6PR. At least 100 cells were scored for compact or dispersed localization. Error bars indicate standard deviations. Similar results were obtained in 3 independent experiments. Significance was calculated employing a two-tailed Student's t test with the Welch correction. *, P ≤ 0.05; **, P ≤ 0.01.
FIG 3
FIG 3
Characterization of a pentapeptide insertion mutant of ppUL35. (A) 293T cells were transfected with expression plasmids for the ppUL35 pentapeptide insertion mutant Tn71 or Tn72 as well as FLAG-SNX5. After 24 h, cell extracts were precipitated with mouse monoclonal antibody against the FLAG (M2) epitope. Precipitated proteins were separated by polyacrylamide gel electrophoresis, and coprecipitated UL35 proteins were detected with a specific rabbit antiserum. Expression of the respective proteins in the cell extracts (lysate controls) was controlled by immunoblotting (bottom). (B) 293T cells were transfected with expression plasmids for wild-type ppUL35 or the Tn71 mutant as well as ppUL82. After 24 h, cell extracts were precipitated with mouse monoclonal antibodies against the ppUL82 (CMV355) or Myc (9E10) epitope. Precipitated proteins were separated by polyacrylamide gel electrophoresis, and coprecipitated UL35 proteins were detected with a specific rabbit antiserum. (C) U373 cells were transfected in triplicate with the HCMV modulator/enhancer luciferase reporter plasmid pHM287 and effector plasmids expressing the indicated genes. All samples contained the same total amount of DNA. After 24 h, luciferase activity was measured. Results are representative of those from 2 or 3 independent experiments. Significance was calculated employing two-tailed Student's t test. *, P ≤ 0.05; ***, P ≤ 0.001; n.s., not significant. (D) HeLa cells were transfected with empty vector as a control or the expression plasmids for wild-type ppUL35 or the ppUL35-Tn71 mutant. At 24 h after transfection, cells were fixed with paraformaldehyde and stained with a rabbit serum directed against ppUL35 and a mouse monoclonal antibody directed against CI-M6PR to assess the subcellular distribution pattern. Two additional experiments yielded comparable results.
FIG 4
FIG 4
Characterization of HCMV mutants. (A) Generation of mutations in the context of the cloned wild-type genome TB40E-BAC4. The UL35 ORF is shown in gray, with the start codons of ppUL35 and ppUL35A indicated by arrows. Mutation of the UL35 start codon was achieved by en passant mutagenesis (TB4-mUL35-M1A), and a revertant (TB4-mUL35-rev; not shown) was also constructed. Other mutants were generated via marker replacement. First, a marker cassette with kanamycin resistance (black square) and an I-SceI site (white triangle) was inserted into the UL35 ORF (TB4-UL35-kan-in). In a second step, the marker cassette was replaced by wild-type UL35 (TB4-UL35-kan-in-rev; not shown), a triple mutant abolishing UL35A translation (TB4-mUL35A), or the pentapeptide insertion mutant Tn71 (TB4-mUL35-Tn71; the pentapeptide insertion indicated by a black diamond). (B) HFF cells infected with the indicated viruses were harvested at 96 h postinfection, and cell extracts were subjected to immunoblotting. Rabbit antisera directed against ppUL35 or ppUL82 or mouse monoclonal antibodies directed against glycoprotein gB (27-287) or β-actin (13E5) were used to assess protein expression. The numbers on the right are molecular mass markers (in kilodaltons). (C) HFF cells were infected with the indicated viruses at an MOI of 1 (left and right) or an MOI of 0.01 (middle). The supernatant was harvested at the indicated time points and titrated on HFF cells.
FIG 5
FIG 5
Localization of glycoprotein B in infected fibroblasts. HFF cells were infected with wild-type or mutant HCMV, as indicated. Cells were fixed with paraformaldehyde at 96 h postinfection and stained (red) for glycoproteins gB (monoclonal 27-287), gH (monoclonal 14-4b), gM (monoclonal IMP91-3/1), and gN (monoclonal 14-16A) (A) or tegument proteins ppUL32 (rabbit serum XP1), ppUL83 (monoclonal 28-77), and ppUL82 (monoclonal CMV355) and suitable secondary antibodies (B). Nuclear counterstaining was achieved using DAPI (blue).
FIG 6
FIG 6
Effect of SNX5 overexpression and depletion. (A) HFF cells were transfected with an expression plasmid for FLAG-SNX5 or control plasmid and infected with TB40E-BAC4 24 h later. At 7 dpi, supernatants were harvested and the virus titers were determined. Three independent experiments were performed, and significance was calculated employing a two-tailed Student's t test. (B) HFF cells were transfected with control or SNX5 siRNA. Cell lysates were analyzed by immunoblotting with staining with polyclonal rabbit anti-SNX5 serum. Immunoblotting with antitubulin served as a loading control. (C) HFF cells transfected with control or SNX5 siRNA were infected with the indicated HCMV wild-type and mutant viruses 24 h later. Supernatants were harvested at 7 dpi, and the virus titer was determined. Results of a representative experiment (from two independent experiments) performed in triplicate are shown. (D) HFF cells were transfected with SNX5 or control siRNA and infected with the indicated wild-type and mutant HCMV 24 h later. At 96 h postinfection, cells were fixed with paraformaldehyde and stained for glycoprotein B. At least 50 cells were scored for compact or dispersed localization. Four to five independent experiments were performed, and significance was calculated employing a two-tailed Student's t test with the Welch correction. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
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