Strain-dependent structural variants of herpes simplex virus type 1 ICP34.5 determine viral plaque size, efficiency of glycoprotein processing, and viral release and neuroinvasive disease potential

Abstract

The ability of certain strains of herpes simplex virus type 1 (HSV-1) to cause encephalitis or neuroinvasive disease in the mouse upon peripheral infection is dependent on a combination of activities of specific forms of viral proteins. The importance of specific variants of ICP34.5 to neuroinvasive disease potential and its correlation with small-plaque production, inefficient glycoprotein processing, and virus release were suggested by comparison of ICP34.5 from the SP7 virus, originally obtained from the brain of a neonate with disseminated disease, and the tissue culture-passaged progeny of SP7 (SLP5 and SLP10) and the KOS321 virus. SLP5, SLP10, and KOS321 are attenuated and exhibit a large-plaque phenotype, including efficient glycoprotein processing and viral release. We show that expression of the KOS321 ICP34.5 protein in cells infected with SP7 or ICP34.5 deletion mutants promotes large plaque formation and efficient viral glycoprotein processing, while expression of the SP7 ICP34.5 protein decreases efficiency of viral glycoprotein processing. In addition, a recombinant virus, 4hS1, with the SP7 ICP34.5 gene replacing the KOS321-like ICP34.5 gene in the SLP10a background, rescues the small-plaque phenotype and neuroinvasive disease. The major difference in the ICP34.5 gene product is the number of Pro-Ala-Thr repeats in the middle region of the protein, with 18 for SP7 and 3 for KOS321. Strain-dependent differences in the ICP34.5 protein can therefore alter the tissue culture behavior and the virulence of HSV-1.

FIG. 1.

Change in the tissue culture behavior upon passage of SP7 leads to the generation of SLP10a. (A) Average plaque size (n = ∼20) of virus from SP7 passages 1 to 10 in Vero cells. A large plaque was picked from passage 10 from which virus stocks for SLP10a were prepared. SLP10a has a KOS321-like ICP34.5 gene (see reference 7). (B) HSV-1 gC from Vero cells infected with SP7, KOS321, or virus from each passage of SP7. Whole-cell detergent extracts from Vero cells infected with each virus were examined by SDS-PAGE and Western blot analysis using polyclonal anti-gC. The image was developed by chemiluminescence and digitally scanned. The black arrow indicates the precursor gC with an apparent mass of 84 kDa, while the open arrow represents the mature gC, which is 116 kDa in mass. The ratio of the amount of the processed gC versus precursor gC is indicated below each lane. PCR analysis indicated a deletion within the ICP34.5 gene for passages 9 and 10.

FIG. 2.

Ectopic expression of ICP34.5-myc variants determines the tissue culture behaviors of an infecting virus. Vero cells were transfected, and 2 days later they were treated with 1 mg of G418/ml for 3 days to inhibit protein synthesis in untransfected cells and then infected with ICP34.5 deletion mutants (d34.5 or TermA) or wild-type viruses (McKrae or AR, a rescue virus from TermA) and harvested 2 days later. Detergent extracts were analyzed by Western blotting. The solid arrows indicate the precursor gC, with an apparent mass of 84 kDa, while the open arrows represent the mature gC, which is 116 kDa. The ratio of the amount of the processed gC to that of precursor gC is indicated below each lane.

FIG. 3.

Ectopic expression of ICP34.5-GFP variants determines the tissue culture behaviors of an infecting virus. Vero cells expressing GFP from the vector or vector containing one of the variants of ICP34.5-GFP were selected based on the expression of GFP using fluorescence-activated cell sorter analysis. The cells were infected with the indicated strain of HSV-1, and gC was analyzed as described in the legend to Fig. 1. The transfecting ICP34.5 variant DNA and the infecting HSV-1 strains are indicated below the image. The solid arrow indicates the precursor gC, with an apparent mass of 84 kDa, while the open arrow represents the mature gC, of 116 kDa. The ratio of the amount of the processed gC to that of precursor gC is indicated below the image.

FIG. 4.

Construction of recombinant virus 4hS1 with an SP7 ICP34.5 gene in an SLP10a background. (A) SLP10a genomic DNA was cotransfected with the ICP34.5-containing BamHI fragment of SP7. The progeny viruses were inoculated into footpads of mice. The lumbrosacral DRGs were collected, and two types of viruses were identified, one with an SP7-like ICP34.5 gene (4hS1) and the other with a KOS321-like gene (4ds). (B) PCR products of the ICP34.5 gene from the different strains were analyzed by electrophoresis. Positions of size standards are indicated in base pairs. LP5 is a clinical strain obtained from the lung of the same neonate as SP7. The LP5 ICP34.5 gene has 22 nucleotide repeats encoding PAT (7).

FIG. 5.

Tissue culture behavior of recombinant virus 4hS1 with an SP7 ICP34.5 gene in an SLP10a background. (A) Average plaque size (n = ∼20) of virus grown in Vero cells. (B) HSV-1 gC from infected Vero cells. Whole-cell detergent extracts from Vero cells infected with each virus were examined by SDS-PAGE and Western blot analysis using polyclonal anti-gC. The image was developed by chemiluminescence and digitally scanned. The black arrow indicates the precursor gC, with an apparent mass of 84 kDa, while the open arrow represents the mature gC, which is 116 kDa. The ratio of the amount of the processed gC to that of precursor gC is indicated below each lane.