Phosphorylation of the herpes simplex virus tegument protein VP22 has no effect on incorporation of VP22 into the virus but is involved in optimal expression and virion packaging of ICP0

Abstract

Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Delta22 virus replicated poorly in epithelial MDBK cells. We have also previously shown that VP22 interacts with the tegument protein VP16 and the cellular microtubule network. While the majority of VP22 in infected cells is highly phosphorylated, the nonphosphorylated form of VP22 is the predominant species in the virion, suggesting a differential requirement for phosphorylation through virus replication. Hence, to study the significance of VP22 phosphorylation, we have now constructed two recombinant viruses expressing green fluorescent protein-VP22 (G22) in which the previously identified serine phosphorylation sites have been mutated either to alanine to abolish the phosphorylation status of VP22 (G22P-) or to glutamic acid to mimic permanent phosphorylation (G22P+). Localization studies indicated that the G22P- protein associated tightly with microtubules in some infected cells, suggesting that VP22 phosphorylation may control its interaction with the microtubule network. By contrast, VP22 phosphorylation had no effect on its ability to interact with VP16 and, importantly, had no effect on the relative packaging of VP22. Intriguingly, virion packaging of ICP0 was reduced in the G22P+ virus while ICP0 expression was reduced in the G22P- virus, suggesting that these two ICP0 defects, previously observed in the Delta22 virus, were attributable to different forms of VP22. Furthermore, the Delta22 virus replication defect in MDBK cells correlated with the expression of constitutively charged VP22 in the G22P+ virus. Taken together, these results suggest an important role for VP22 phosphorylation in its relationship with ICP0.

FIG. 1.

Phosphorylation variants of VP22 as GFP-VP22 fusion proteins. (A) Line drawing of the GFP-VP22 (G22) open reading frame showing GFP (black box) and the 301 amino acids of the VP22 WT (white box). The serine (S)-to-alanine (A) or S-to-glutamic acid (E) substitutions present in the plasmids expressing the GFP-VP22P− (G22P−) or GFP-VP22P+ (G22P+) mutant are shown. These substitutions were introduced by PCR mutagenesis. (B) Live transfected Cos-1 cells expressing either the G22, G22P−, or G22P+ proteins, examined by confocal microscopy 24 h posttransfection.

FIG. 2.

Characterization of VP22 expressed in G22, G22P− or G22P+ virus infections. (A) Monolayers of Vero cells were infected with G22v, G22P−v, or G22P+v at a multiplicity of infection of 10, and total cell lysates were harvested after 10 h. Equal amounts of lysate were analyzed by SDS-PAGE followed by Western blotting with an antibody against VP22. (B) Vero cells were infected at a multiplicity of infection of 10 with the WT viral s17, GFP-VP22 expressing G22v, or the recombinant viruses G22P−v and G22P+v and incubated in a labeling medium containing [³²P]orthophosphate. Protein cell lysates were immunoprecipitated using an antibody directed against VP22 and then analyzed by Western blotting using an antibody directed against GFP. (C) SDS-PAGE analysis of the immunoprecipitated proteins and autoradiography. The positions of VP22 (➤) and GFP-VP22 (*) are shown.

FIG. 3.

Live-cell analysis of the VP22 phosphorylation variants in infected cells. Monolayers of Vero cells grown in coverslip chambers were infected with G22v (A, D, G, and J), G22P−v (B, E, H, and K), or G22P+v (C, F, I, and L) at a multiplicity of infection of 0.1 and were examined by confocal microscopy for GFP fluorescence every 2 h up to 20 h postinfection (h.p.i.). Representative images taken at 8 (A to C), 12 (Dto F), 16 (G-I), or 20 (J to L) h.p.i. are shown.

FIG. 4.

The G22P− protein forms MT bundles in infected cells. Vero cells infected with G22P-v at a multiplicity of infection of 0.1 were examined live at 20 h postinfection (A) or were fixed with methanol and processed for immunofluorescence with an anti-α-tubulin antibody. (B) Cells were examined by confocal microscopy for GFP-VP22 (green) and α-tubulin fluorescence (red). Right-hand panels show magnified images of the region in the white box.

FIG. 5.

The phosphorylation variants of VP22 interact with VP16. (A) Monolayers of Vero cells were either mock infected or infected at a multiplicity of infection of 10 with G22v, G22P−v, G22P+v, or the Δ22 virus (169v) and lysed in radioimmunoprecipitation assay buffer 15 h postinfection. Cell lysates were immunoprecipitated using an antibody directed against VP22 and then analyzed by SDS-PAGE and Western blotting with antibodies against GFP and VP16. (B) Monolayers of Vero cells were infected with either G22v or G22P−v at a multiplicity of infection of 0.1. At 20 h postinfection, the cells were fixed with methanol and processed for immunofluorescence with an anti-VP16 antibody. Cells were examined by confocal microscopy for GFP-VP22 (green) and VP16 (red) fluorescence.

FIG. 6.

ICP0 expression and localization in cells infected with the VP22 phosphorylation mutants. (A) Monolayers of Vero cells were infected with G22v, G22P−v, or G22P+v at a multiplicity of infection of 10, and total cell lysates were harvested every 5 h after infection up to 20 h. Equal amounts of total cell lysates were analyzed by SDS-PAGE followed by Western blotting with antibodies against GFP, ICP4, ICP0, VP16, gD, and cellular β-actin. (B) Monolayers of Vero cells were infected with G22v, G22P−v, or G22P+v at a multiplicity of infection of 10 and fixed 7 h later in 4% paraformaldehyde. The cells were then processed for immunofluorescence with a monoclonal anti-ICP0 antibody and examined by confocal microscopy for GFP-VP22 (green) and ICP0 fluorescence (red). Note that the ICP0 image for G22P−v was imaged using 10-fold more laser power because of its weak expression in infected cells.

FIG. 7.

Incorporation of the phosphorylation variants of VP22 into virions. Approximately equivalent amounts of purified G22v, G22P−v, and G22P+v virions were solubilized and analyzed by SDS-PAGE followed by either Coomassie blue staining (A) or Western blotting with antibodies directed against VP22, VP16, gD, ICP0, and ICP4 (B). (C) Equivalent amounts of purifed G22v, G22P−v, and three different preparations of G22P+v virions were analyzed by Western blotting for ICP0 and VP5.

FIG. 8.

HSV-1 expressing the P+ version of VP22 displays a growth defect in MDBK cells. (A) Monolayers of MDBK cells were infected with WT, Δ22 G22P−, or G22P+ viruses at a multiplicity of infection of 0.02. At various times after infection, total virus was harvested by combining the cells and medium and the samples were titrated on Vero cells. (B) Monolayers of MDBK cells were infected with Δ22, G22, G22P−, or G22P+ viruses at a multiplicity of infection of 10. At various times after infection, total cell extracts were made from infected cells and analyzed by Western blotting for VP22 and ICP0.