Glycoprotein K specified by herpes simplex virus type 1 is expressed on virions as a Golgi complex-dependent glycosylated species and functions in virion entry

Abstract

To facilitate detection of glycoprotein K (gK) specified by herpes simplex virus, a 12-amino-acid epitope tag was inserted within gK domain III. Recombinant virus gKprotC-DIII, expressing the tagged gK, was isolated. This virus formed wild-type plaques and replicated as efficiently as the wild-type KOS virus in Vero cells. Anti-protein C MAb detected high-mannose and Golgi complex-dependent glycosylated gK within cells as well as on purified virions. The gK-null virus DeltagK (gK(-/-)) entered Vero cells substantially more slowly than the wild-type KOS (gK(+/+)), while DeltagK virus grown in complementing VK302 cells (gK(-/+)) entered with entry kinetics similar to those of the KOS virus.

FIG. 1

Construction of recombinant virus gKprotC-DIII, specifying gK containing a protein C epitope tag. (A) The top line represents the prototypic arrangement of the HSV-1 genome with the unique long (U_L) and unique short (U_S) regions flanked by the terminal repeat (TR) and internal repeat (IR) regions. (B) Shown below is the region of the mutant virus HSV-1 d27-1 genome (between map units 0.7 and 0.8) containing the UL52, UL53, and the partially deleted UL54 open reading frames (shaded and boxed regions of the UL54 gene pointed to by an arrow) with relevant restriction endonuclease sites. (C) Plasmid construct, pTF9301, containing the recombinant gK-protC gene and flanking UL52 and UL54 sequences used to generate recombinant virus gKprotC-DIII. (D) Schematic model of the predicted secondary structure of gK (10). The predicted putative hydrophobic domains (hpd) of gK that transverse the membrane are shown embedded within the membrane. The arrow points to the site of the protC epitope tag insertion. The primary structure of the epitope tag is shown. Known syncytial mutations are denoted by asterisks.

FIG. 2

Comparison of plaque morphology and replication characteristics of gKprotC-DIII and KOS viruses. Comparison of virus plaque morphologies formed on Vero cells at 48 h postinfection. (A) KOS. (B) gKprotC-DIII. (C) Time-dependent kinetics of infectious virus production after infection of Vero cells at an MOI of 5 and incubation at 37°C. The graph depicts one of three separate experiments with similar results. Each separate experiment was repeated in triplicate to obtain standard deviations.

FIG. 3

Characterization of synthesis and processing of protC-tagged gK specified by gKprotC-DIII. Immunoblots of gKprotC-DIII- (lanes 1 to 3) and KOS (lanes 4 to 6)-infected cell extracts reacted with either anti-protC MAb HPC-4 (A) or anti-gD MAb 1103 (B). Cellular extracts were treated with Endo-H (lanes 2 and 5), PNGase-F (lanes 3 and 6), or mock treated (lanes 1 and 4). (C) Cellular extracts obtained from Vero cells infected with gKprotC-DIII in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of TM were probed with either anti-protC MAb (lanes 1 and 2) or anti-gD MAb (lanes 3 and 4).

FIG. 4

Detection and characterization of protC-tagged gK expressed on purified virions. Immunoblots of gKprotC-DIII (lanes 1 to 3) or KOS (lanes 4 to 6) purified virion preparations reacted with anti-protC MAb HPC-4 (A), anti-gD MAb 1103 (B), and anti-ICP27 MAb 1113 (C). Purified virion preparations were treated with Endo-H (lanes 2 and 5), PNGase-F (lanes 3 and 6), or mock treated (lanes 1 and 4). (D) Cellular extracts from Vero cells infected with either gKprotC-DIII (lanes 1 to 3) or KOS (lanes 4 to 6) were reacted with anti-ICP27 MAb. Cellular extracts were treated with Endo-H (lanes 2 and 5), PNGase-F (lanes 3 and 6), or mock treated (lanes 1 and 4).

FIG. 5

Penetration kinetics of KOS and ΔgK viruses into VK302 (Vero) cells. The penetration kinetics of KOS virus grown on Vero cells and ΔgK virus grown on either Vero or VK302 cells were obtained by determining the percentage of PFU surviving low-pH treatment relative to PBS-treated controls at different times postadsorption. Mean values and standard deviations of three independent experiments are shown.