Characterization of a UL49-null mutant: VP22 of herpes simplex virus type 1 facilitates viral spread in cultured cells and the mouse cornea

Abstract

Herpes simplex virus type 1 (HSV-1) virions, like those of all herpesviruses, contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The HSV-1 tegument is composed of at least 20 different viral proteins of various stoichiometries. VP22, the product of the U(L)49 gene, is one of the most abundant tegument proteins and is conserved among the alphaherpesviruses. Although a number of interesting biological properties have been attributed to VP22, its role in HSV-1 infection is not well understood. In the present study we have generated both a U(L)49-null virus and its genetic repair and characterized their growth in both cultured cells and the mouse cornea. While single-step growth analyses indicated that VP22 is dispensable for virus replication at high multiplicities of infection (MOIs), analyses of plaque morphology and intra- and extracellular multistep growth identified a role for VP22 in viral spread during HSV-1 infection at low MOIs. Specifically, VP22 was not required for either virion infectivity or cell-cell spread but was required for accumulation of extracellular virus to wild-type levels. We found that the absence of VP22 also affected virion composition. Intracellular virions generated by the U(L)49-null virus contained reduced amounts of ICP0 and glycoproteins E and D compared to those generated by the wild-type and U(L)49-repaired viruses. In addition, viral spread in the mouse cornea was significantly reduced upon infection with the U(L)49-null virus compared to infection with the wild-type and U(L)49-repaired viruses, identifying a role for VP22 in viral spread in vivo as well as in vitro.

FIG. 1.

Genetic analysis of WT, U_L49⁻, and U_L49R viral DNAs. (A) Schematic representation of the BamHI F fragment in WT and U_L49R (upper) and U_L49⁻ (lower) viral genomes. Replacement of the U_L49 gene with a FRT-BamHI cassette in the U_L49⁻ viral genome yielded 6,512-bp and 724-bp BamHI fragments in place of the 8,054-bp BamHI F-fragment present in the WT and U_L49R viral genomes. (B) Negative scanned image of an agarose gel containing BamHI-digested WT, U_L49⁻, and U_L49R viral DNAs visualized with ethidium bromide staining. (C) Scanned autoradiograph of Southern DNA hybridization of the same gel in which [α-³²P]dCTP-labeled HSV-1 BamHI F-fragments were used as probes.

FIG. 2.

Immunoblot analysis of VP16 expression upon infection with the WT, U_L49⁻, and U_L49R viruses. Lysates of Vero cells infected with the WT, U_L49⁻, or U_L49R viruses for 18 h were separated by SDS-PAGE and probed for the presence of VP22 (top) and VP16 (middle). As a loading control, the immunoblot was also probed with an antibody directed against pU_L28 (bottom).

FIG. 3.

Analysis of U_L49.5 expression upon infection with the WT, U_L49⁻, and U_L49R viruses. (A and B) Genetic analysis of U_L49.5⁻ viral DNA. (A) Deletion of nucleotides encoding U_L49.5 residues 9 to 22 in the U_L49.5⁻ viral genome yielded a 5,371-bp EcoRV fragment (○) in place of the 5,553-bp EcoRV fragment (*) present in the WT viral genome. (B) One-percent agarose gel electrophoresis of EcoRV-digested WT and U_L49.5⁻ viral DNAs visualized with ethidium bromide staining (negative image, left) and Southern hybridization (right) using the ApoI-BamHI segment of the HSV-1 BamHI F-fragment labeled with [α-³²P]dCTP as a probe. (C) Scanned autoradiographic image of a 16% Tricine-SDS-polyacrylamide gel containing electrophoretically separated [³⁵S]methionine-cysteine-labeled proteins from lysates of cells infected with WT, U_L49⁻, U_L49R, and U_L49.5⁻ viruses. The location of the U_L49.5 gene product in the WT, U_L49⁻, and U_L49R lanes is indicated.

FIG. 4.

Analysis of plaques produced by the WT, U_L49⁻, and U_L49R viruses in cultured cells. Vero cell monolayers were infected with the WT, U_L49⁻, or U_L49R viruses at an MOI of ∼1 × 10⁻⁵ PFU/cell for 44 h. Cells were then fixed and prepared for immunofluorescence microscopy using an antibody directed against the viral protein gM. Plaques were visualized under a fluorescence microscope and photographed with a digital camera. (A) Representative photographs of WT, U_L49⁻, and U_L49R viral plaques. Scale bar, 500 μm. (B) Mean areas of 70 U_L49⁻ and 70 U_L49R plaques relative to the mean area of 70 WT plaques. Error bars represent 1 standard deviation.

FIG. 5.

Single-step growth analyses of the WT, U_L49⁻, and U_L49R viruses. Vero cell monolayers were infected with the WT, U_L49⁻, and U_L49R viruses at an MOI of 5 PFU/cell for 1 h to allow virus adsorption. The cells were then washed extensively with citrate buffer to neutralize and remove unbound virus. The cells were overlaid with medium and held at 37°C. At the indicated times postinfection, the infected cells (A) and the overlaying medium (B) were analyzed separately by plaque assay to determine intracellular and extracellular viral yields, respectively.

FIG. 6.

Multistep growth analyses of the WT, U_L49⁻, and U_L49R viruses in the presence and absence of neutralizing antibody. Vero cell monolayers were infected with the WT, U_L49⁻, and U_L49R viruses at an MOI of 0.001 PFU/cell for 1 h to allow virus adsorption. The cells were then washed extensively with citrate buffer to neutralize and remove unbound virus, overlaid with medium, and held at 37°C. Cells infected for intracellular multistep growth analysis in the presence of neutralizing antibody were overlaid with medium containing 0.3% human gamma globulins. At the indicated times postinfection, these cells were pelleted, washed three times with PBS to remove the neutralizing antibody, and lysed. Also at the indicated times postinfection, medium was removed from cells grown in the absence of neutralizing antibody and clarified. Intracellular virus was titrated from the cell lysates and extracellular virus was titrated from the clarified medium. The growth curves shown represent the means and standard deviations (error bars) of three independent experiments. Growth curves in the upper panels were performed at different times using different passages of Vero cells than those of the lower panel. Therefore, titers between upper and lower panels are not comparable.

FIG. 7.

Analysis of extracellular WT, U_L49⁻, and U_L49R virions. Extracellular virions were purified from the clarified medium overlying cells infected with either the WT, U_L49⁻, or U_L49R viruses by gradient centrifugation. (A) The number of PFU/ml in gradient fractions was determined by plaque assay on Vero cells. Fraction 1 represents the bottom of the gradient, and fraction 9 represents the top of the gradient. (B) Immunoblots of the same gradient fractions probed with an antibody against the major capsid protein VP5. Each lane is aligned with the corresponding fraction in panel A.

FIG. 8.

Analysis of the protein composition of WT, U_L49⁻, and U_L49R virions. Intracellular virions were purified from cells infected with either the WT, U_L49⁻, or U_L49R viruses. Virions proteins were electrophoretically separated on a 12% SDS-polyacrylamide gel and visualized by either Coomassie staining (left panel) or immunoblot analyses using antibodies against VP5, ICP0, gE, VP16, gD, β-actin, and VP22 (right panels). The immunoblots are aligned with the position of each probed protein in the Coomassie-stained gel.

FIG. 9.

Analysis of U_L49⁻, U_L49R, and WT HSV-1 viral spread in the mouse cornea. The corneas of live mice were scarified and infected with ∼1,000 PFU of either U_L49⁻ (A to C), U_L49R (D to F), or WT HSV-1 (G to I) virus. At 24 or 48 hpi, the mice were sacrificed, and the corneas were dissected and prepared for immunohistochemistry using an antibody directed against HSV-1 proteins. Photographs of corneas flat mounted on glass slides are shown with the left and middle panels at the same magnification (bar = 100 μm) and at lower magnification in the right panels (bar = 1 mm).