Deletion of the herpes simplex virus VP22-encoding gene (UL49) alters the expression, localization, and virion incorporation of ICP0

Abstract

The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Delta22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Delta22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Delta22-infected cells correlates with an absence of both ICP0 and ICP4 from Delta22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly.

FIG. 1.

Characterization of an inducible VP22-expressing 293 cell line. (A) The VP22-293-EcR cell line was induced to express VP22 with 1 μg/ml muristerone A, and samples were harvested at various times after induction and analyzed by SDS-PAGE and Western blotting with antibodies against VP22 and α-tubulin. (B) VP22-293-EcR cells grown on coverslips were left uninduced or induced with muristerone A, fixed 24 h later, and stained with polyclonal anti-VP22. Arrows indicate cells with VP22 in the nucleus; arrowheads indicate cells with VP22 in the cytoplasm. -ve, parental 293-EcR cell line.

FIG. 2.

Characterization of the Δ22 virus. (A) DNA map of the HSV-1 genome across the UL49 gene, showing the relevant BamHI (Ba) and EcoRV (RV) sites. (B) Southern blotting of EcoRV- and BamHI-digested genomic DNA from WT and Δ22 viruses. The blots have been hybridized with probes for the UL49, GFP, and UL47 genes. (C) Monolayers of Vero cells were infected with either WT or Δ22 virus at a multiplicity of 10 and harvested at various time points ranging from 5 to 25 h. Equal amounts of total cell lysates were analyzed by SDS-PAGE followed by Western blotting with antibodies against VP22, GFP, and α-tubulin. Numbers to the left indicate DNA size markers in kilobases.

FIG. 3.

Characterization of HSV-1 virions assembled in the absence of VP22. (A) Equivalent amounts of purified extracellular WT and Δ22 virions were analyzed by SDS-PAGE and stained with Coomassie blue. Major virion proteins are labeled. The arrowhead denotes a species around 175 kDa that is absent from Δ22 virions. Numbers on the left indicate molecular mass in kilodaltons. (B) The same samples shown in panel A were analyzed by Western blotting with antibodies against the indicated virion proteins. (C) Monolayers of Vero cells were infected at 4°C with approximately 300 PFU of virus stock. At the indicated times, the cells were washed and transferred to 37°C, and plaques were allowed to develop. (D) Monolayers of Vero cells were infected with approximately 300 PFU of virus stock and incubated at 4°C for 2 h. Cells were transferred to 37°C and washed with citrate buffer (pH 3) at the indicated times, and plaques were allowed to develop.

FIG. 4.

The Δ22 virus exhibits an early delay in the infectious process. (A) Monolayers of Vero cells were infected with either WT or Δ22 virus stocks at a multiplicity of 10 followed by pulse-labeling with [³⁵S]methionine at various times after infection. The cells were harvested and analyzed by SDS-PAGE and autoradiography. Asterisks indicate virus-induced proteins. Numbers on the left indicate molecular masses in kilodaltons. (B) Monolayers of Vero cells were infected with the same WT or Δ22 virus stocks at a multiplicity of 10, and samples were harvested at the indicated times after infection and analyzed by SDS-PAGE and Western blotting with antibodies against virus proteins as indicated. (C) Monolayers of Vero cells were infected with WT, Δ22, or Δ22R virus at a multiplicity of 10, and samples were harvested at the indicated times after infection and analyzed by SDS-PAGE and Western blotting with antibodies against VP22 and ICP0. h.p.i., hours postinfection.

FIG. 5.

The Δ22 virus exhibits a replication defect in MDBK cells compared to Vero cells. (A) Monolayers of Vero cells were infected with WT or Δ22 virus at a multiplicity of 0.1. 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 WT, Δ22, or Δ22R virus at a multiplicity 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. h.p.i., hours postinfection.

FIG. 6.

Localization of ICP0 in Δ22 virus-infected cells. MDBK and BHK cells grown on coverslips were infected with either WT virus or Δ22 virus at a multiplicity of 10. At 2, 4, and 8 h after infection, the cells were fixed and processed for immunofluorescence with the anti-ICP0 antibody 11060.

FIG. 7.

VP22 colocalizes with ICP0 and is required for ICP0 incorporation into virions. (A) Vero, MDBK, and BHK cells grown on coverslips were infected with the GFP-VP22-expressing virus 166v at a multiplicity of 10. At 6 h, the cells were fixed and processed for immunofluorescence with the anti-ICP0 antibody 11060. The insets show magnified images of the indicated regions. (B) Virion samples of WT, Δ22, and Δ22R viruses grown in BHK21 cells were analyzed by SDS-PAGE followed by Western blotting with antibodies against VP5, ICP0, ICP4, and VP22.