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, 89 (19), 9841-52

Cellular Protein WDR11 Interacts With Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

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Cellular Protein WDR11 Interacts With Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

Kathryne E Taylor et al. J Virol.

Abstract

It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment.

Importance: While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both our understanding of basic cellular biology as well as how this protein is co-opted by HSV.

Figures

FIG 1
FIG 1
ICP0 interacts with WDR11 but does not cause its degradation. (A) HEL cells were infected with the indicated viruses at 10 PFU/cell for 8 h, and then samples were harvested via cytoplasmic extract and immunoprecipitated with the indicated antibodies. Eluents were then analyzed via Western blotting for ICP0 and WDR11. (B) HEL cells were infected with the indicated viruses at 10 PFU/cell for 10 h and then harvested in RIPA extract and analyzed for WDR11, ICP0, and actin levels via Western blotting.
FIG 2
FIG 2
WDR11 has a distinct perinuclear distribution that becomes dispersed at late times after HSV-1 infection in an ICP0-independent manner. HEL cells (A) or U2OS cells (B) were mock treated or infected with the indicated viruses at 10 PFU/cell for the times shown and then fixed and analyzed for WDR11 localization via immunofluorescence. Nuclei were identified using Hoechst stain.
FIG 3
FIG 3
WDR11 relocalization is specific to HSV infection and requires viral late gene expression. (A) HEL cells were infected with 17 syn (10 PFU/cell), HCMV (0.5 PFU/cell), SeV (80 HAU/106 cells), or VSV (0.1 PFU/cell) or treated with 100 μg/ml pI·C for 16 h. Infected cells were then fixed and analyzed for WDR11 localization via immunofluorescence. (B) HEL cells were mock treated or infected with 17 syn at 10 PFU/cell for 10 h in the presence or absence of 400 μg/ml PAA and then analyzed for WDR11 localization and gC expression via immunofluorescence. Nuclei were identified using Hoechst stain.
FIG 4
FIG 4
WDR11 colocalizes with the TGN marker TGN46. HEL cells were mock treated (A), infected with 17 syn at 10 PFU/cell for 10 h (B), or treated with 10 μg/ml BFA or the vehicle control for 30 min (C) and then fixed and analyzed for TGN46 and WDR11 localization via immunofluorescence.
FIG 5
FIG 5
Interaction of specific additional HSV proteins with WDR11. (A and B) HEL cells (A) or U2OS cells (B) were mock treated or infected with the indicated viruses at 10 PFU/cell for 10 h. (C) U2OS cells were transfected with either the empty vector (EV) or a plasmid carrying a mutant ICP0 expressed in the cytoplasm (D8) for 24 h. In all three cases, cytoplasmic extracts were then harvested, immunoprecipitations were performed with an anti-WDR11 antibody, and eluents were analyzed via Western blotting with the indicated antibodies.
FIG 6
FIG 6
WDR11 depletion reduces yields of both cell-associated and secreted HSV-1 without decreasing viral gene expression. U2OS cells were treated with WDR11 or nontargeting control siRNA for 72 h. (A) Cells on coverslips were infected with 17 syn at 10 PFU/cell for 10 h and then fixed and analyzed for WDR11 levels via immunofluorescence. Nuclei were identified using Hoechst stain. (B) Cells were infected with 17 syn at 10 PFU/cell for 10 h, and then RIPA extracts were harvested and analyzed for the expression of the indicated proteins via Western blotting. (C) Cells were infected with the indicated viruses at 10 PFU/cell for 24 h. Cells and supernatant medium were then harvested separately, the cell-associated fraction was freeze-thawed three times, and then titers in both fractions were determined on U2OS cells in the presence of HMBA. The data are averages of 3 independent replicates ± standard errors of the means. Statistical analysis was performed using one-way analysis of variance and Bonferroni's multiple-comparison posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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