The amino terminus of herpes simplex virus type 1 glycoprotein K (gK) modulates gB-mediated virus-induced cell fusion and virion egress

Abstract

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKDelta31-47, gKDelta31-68, and gKDelta31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKDelta31-47 and gKDelta31-117 exhibited a gK-null (DeltagK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKDelta31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKDelta31-47 and gKDelta31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBDelta28syn) caused extensive virus-induced cell fusion. However, the gBDelta28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKDelta31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKDelta31-68, DeltagK, or UL20-null virus. The gKa peptide complemented the gKDelta31-47 and gKDelta31-68 mutant viruses for infectious-virus production and for gKDelta31-68/gBDelta28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.

FIG. 1.

HSV-1 gK membrane topology. (A) The experimentally validated topology of gK is shown in conjunction with the secondary predicted structure of gK (18). Features of the gK topology include four membrane-spanning hydrophobic domains (hpd1 to -4), a signal sequence of 30 aa, and two N-linked glycosylation sites (CHO). The locations of different syncytial mutations published previously (17, 18) are indicated by stars. (B) Schematic showing the in-frame truncations within the amino terminus of gK demarcated with dotted lines spanning aa 31 to 47, 31 to 68, and 31 to 117. Similar dotted markings are also included in panel A. The gene construct encoding the amino-terminal 82 aa of gK in-frame with the 3× FLAG epitope is shown.

FIG. 2.

Plaque morphologies and relative sizes of gK mutant and wild-type HSV-1(F) viruses. Vero cells were infected with an MOI of 0.001, and viral plaques were fixed with methanol and stained with anti-HSV antibodies as described in Materials and Methods. (A) Representative viral plaques of all gK mutant viruses and the HSV-1(F) wild-type virus are shown on both Vero (A to E) and VK302 (F to J) cells (constitutively expressing gK). (B) Bar graph showing the average plaque sizes of 70 randomly chosen viral plaques for each virus; error bars indicate the standard error (see Materials and Methods).

FIG. 3.

One-step replication kinetics of HSV-1(F) wild-type (wt) and gK mutant viruses. Vero cells were infected at either a low MOI (MOI of 0.1) or a high MOI (MOI of 2), and the numbers of infectious viruses produced were determined on VK302 cells at different times postinfection. At each time point, supernatants and infected cells were collected, and virus titers were determined separately. Viral titers after low-MOI infection are shown in panels A (intracellular) and B (supernatant). High-MOI titers are shown in panels C (intracellular) and D (supernatant). Viral titers obtained after low-MOI infections on VK302 cells are shown in panel E. Ratios of intracellular to extracellular viral titers obtained at 24 hpi are shown in panel F. gKins17, ΔgK recombinant virus created by inserting a kanamycin resistance cassette after aa position 17 within the gK signal sequence.

FIG. 4.

Plaque morphologies of the gBΔ28syn and gKΔ31-68/gBΔ28syn viruses on Vero and VK302 cells. Viral plaques were obtained on both Vero and VK302 cells and visualized after methanol fixing and immunostaining with anti-HSV antibodies at 48 hpi. Magnified parts of viral plaques are shown as insets in each photograph.

FIG. 5.

Transient expression of the gKa peptide. Vero cells were transfected with the gKa-expressing plasmid, and gKa expression was detected by a Western immunoblot assay utilizing the anti-FLAG antibody. Cellular extracts from transfected Vero cells (lane 5) and transfected cells followed by infection with the gBΔ28syn virus (lane 6) are shown. Cellular extracts were treated with either endo H (lane 1, gKa transfection alone; lane 2, gKa transfection followed by gBΔ28syn infection) or PNGase F (lane 3, gKa transfection alone; lane 4, gKa transfection followed by gBΔ28syn infection). Molecular mass markers, shown as dots, are 10, 15, and 20 kDa.

FIG. 6.

Complementation of viral replication of gK mutant viruses by the gKa peptide. Vero cells were transfected with the gKa-expressing plasmid or a control plasmid containing the gKa coding sequence cloned in reverse (see Materials and Methods), and 24 h posttransfection, cells were infected with either the gKΔ31-47 or the gKΔ31-68 virus at an MOI of 1. To better visualize the ∼2-fold complementation for viral replication effect, the numbers of viral plaques obtained at the 10⁻³ dilution for extracellular (A) and combined extracellular and intracellular (B) virus stocks obtained at 21 hpi are shown. Complementation by wild-type gK for total virus (extracellular plus intracellular) at 9 hpi in comparison to complementation by gKa is shown in panel C. Error bars indicate the standard error.

FIG. 7.

Cell surface expression of the gKa peptide. Vero cells were transfected with the gKa plasmid or the control plasmid gKa-R, and transfected cells were infected with the gKΔ31-68/gBΔ28syn virus. Cells were stained with either the anti-FLAG (α-FLAG) or the anti-HSV-1 (α-HSV) antibody at 30 hpi under either methanol-fixed (A) or live (B) conditions. The percentages of FLAG-positive live cells in gKa-transfected cells infected with either wild-type HSV-1(F) or the ΔgK or ΔUL20 mutant are shown as contour plots (C) and as bar graphs (D). SSC, side scatter.

FIG. 8.

Ability of the gKa peptide to complement gKΔ31-68/gBΔ28syn-induced cell fusion. (A) Vero cells were transfected with the gKa, gKa-R, gKa-A40V, or gKa-A40T plasmid, and cells were subsequently infected with the gKΔ31-68/gBΔ28syn virus at an MOI of 0.2. Cells were reacted with the anti-FLAG (α-FLAG) or the anti-HSV (α-HSV) antibody under methanol-fixed conditions and visualized by phase-contrast microscopy after immunostaining. (B) Vero cells were transfected and infected as detailed above, with the exception that two different populations of cells were transfected at the same time, with each of the gK-expressing plasmids with a plasmid expressing T7 polymerase (pol) or a plasmid expressing the luciferase gene under the T7 promoter. These equal populations of cells were mixed together prior to infection with the gKΔ31-68/gBΔ28syn virus. The amount of relative fusion was obtained by measuring the relative level of luminescence (RLU) emitted by cellular extracts at 24 hpi. Error bars indicate the standard error.