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. 1998 Mar;72(3):1949-58.
doi: 10.1128/JVI.72.3.1949-1958.1998.

Pseudorabies virus glycoprotein gK is a virion structural component involved in virus release but is not required for entry

B G Klupp  1 , J Baumeister, P Dietz, H Granzow, T C Mettenleiter
Affiliations

Pseudorabies virus glycoprotein gK is a virion structural component involved in virus release but is not required for entry

B G Klupp et al. J Virol. 1998 Mar.

Abstract

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560-5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase-gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKbeta) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB- genome (PrV-gK(gB)). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKbeta was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.

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Figures

FIG. 1
FIG. 1
Construction of PrV gK mutants, complementing cell lines, and GST-gK fusion protein. (A) Schematic diagram of the PrV genome with the BamHI restriction fragment map. The PrV genome consists of a unique long region (UL) and a unique short region (US). The latter is flanked by inverted repeats (IR, internal repeat; TR, terminal repeat). (B) Enlargement of BamHI fragment 5′. Locations of the identified ORFs with transcriptional orientation, indicated by arrows, are given (R, region of reiterated sequences), and relevant restriction sites are marked, as is the location of fragment Sal2 used for cloning. (C) Part of viral BamHI fragment 5′ introduced into cell line C53/54. (D) Schematic diagram of gK. Indicated in dark grey are predicted signal sequence and transmembrane domains. (E) Prokaryotic expression of gK. In plasmid pGEX-Bst XI/Xho I, a fragment comprising codons 64 to 196 of the gK ORF was cloned downstream from and in frame with the GST gene (not drawn to scale) and expressed in E. coli for immunization of a rabbit.
FIG. 2
FIG. 2
Identification of PrV gK. Proteins of purified PrV-Ka (lanes 1), PrV-gKβ (lanes 2), or PrV-gKgB (lanes 3) virions or lysate of PrV-Ka-infected cells harvested 24 h p.i. (lanes 4) were separated by gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with the PrV gK-specific rabbit polyclonal serum (A), a gH-specific rabbit serum (B), or a dUTPase-specific serum (C). Bound antibody was visualized by chemiluminescence after incubation with a peroxidase-conjugated secondary antibody. Locations of molecular mass markers are indicated on the left.
FIG. 3
FIG. 3
Analysis of N-linked carbohydrates on PrV gK. Purified PrV-Ka virions were incubated either with endo H (lanes 2), with PNGase F (lanes 3), or without enzyme (lanes 4) or were separated without prior treatment (lanes 1). After gel electrophoresis and Western blotting, replica filters were incubated with serum specific for PrV gK (A) or gH (B). Locations of molecular mass markers are indicated on the left.
FIG. 4
FIG. 4
Plaque morphology of PrV gK mutants. Noncomplementing Vero and complementing B5′-64 and C53/54 cells were infected with PrV-1112, PrV-gKβ, and PrV-gKgB under plaque assay conditions. Cells were fixed 2 days p.i., stained with X-Gal, and photographed. Bar = 500 μm.
FIG. 5
FIG. 5
Penetration kinetics of PrV-1112 and PrV-gKβ. Complementing cells were infected with either PrV-1112 or PrV-gKβ grown on noncomplementing or complementing (PrV-gKβc) cells. After 1 h of incubation on ice, cells were overlaid with prewarmed medium to initiate penetration. At different times thereafter, remaining extracellular virus was inactivated by low-pH treatment. The percentage of PFU surviving low-pH treatment was calculated with reference to a PBS-treated control as percent penetration. Mean values and standard deviations of results of at least three independent experiments are shown.
FIG. 6
FIG. 6
One-step growth analysis. Complementing and noncomplementing cells were infected at an MOI of 10 with PrV-Ka or PrV-gKβ (A) or at an MOI of 5 with PrV-9112C2 or PrV-gKgB (B) for 1 h at 4°C. After an additional 2 h at 37°C, remaining extracellular virus was inactivated by low-pH treatment. Immediately thereafter (0 h) and at the time points indicated, cells and supernatant were harvested, and progeny virus was titrated on complementing C53/54 cells. Mean values and standard deviations of results of two independent experiments are shown.
FIG. 7
FIG. 7
Electron microscopy of PrV-gKβ on complementing cells. B5′-64 cells were infected with PrV-gKβ at an MOI of 1 and analyzed 16 h p.i. The arrow shows primary envelopment in the perinuclear cisterna (B), small arrowheads indicate secondary envelopment in the Golgi area (B), and large arrowheads point to free virions in the extracellular space (A to C). Bars: A, 2.5 μm; B, 1 μm; C, 1 μm.
FIG. 8
FIG. 8
Electron microscopy of PrV-gKβ on noncomplementing cells. Noncomplementing Vero cells were infected with PrV-gKβ at an MOI of 1 and analyzed 16 h p.i. The arrow in panel B (inset) indicates primary envelopment by budding into the perinuclear cisterna, small arrowheads in panels B and C point to secondary envelopment in the cytoplasm, and large arrowheads in panels A, D, and E show fusion stages at the cytoplasmic membrane which are typical for PrV gK mutants. Bars: A, 2.5 μm; B, 1 μm; inset, 150 nm; C, 300 nm; D, 1 μm; E, 200 nm.
FIG. 9
FIG. 9
Diagram of proposed events and involvement of viral proteins during egress of PrV.
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