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. 2000 Dec;74(24):11782-91.
doi: 10.1128/jvi.74.24.11782-11791.2000.

Glycoprotein D or J delivered in trans blocks apoptosis in SK-N-SH cells induced by a herpes simplex virus 1 mutant lacking intact genes expressing both glycoproteins

G Zhou  1 , V Galvan, G Campadelli-Fiume, B Roizman
Affiliations

Glycoprotein D or J delivered in trans blocks apoptosis in SK-N-SH cells induced by a herpes simplex virus 1 mutant lacking intact genes expressing both glycoproteins

G Zhou et al. J Virol. 2000 Dec.

Abstract

We have made two stocks of a herpes simplex virus 1 mutant lacking intact U(S)5 and U(S)6 open reading frames encoding glycoproteins J (gJ) and D (gD), respectively. The stock designated gD(-/+), made in cells carrying U(S)6 and expressing gD, was capable of productively infecting cells, whereas the stock designated gD(-/-), made in cells lacking viral DNA sequences, was known to attach but not initiate infection. We report the following. (i) Both stocks of virus induced apoptosis in SK-N-SH cells. Thus, annexin V binding to cell surfaces was detected as early as 8 h after infection. (ii) U(S)5 or U(S)6 cloned into the baculovirus under the human cytomegalovirus immediate-early promoter was expressed in SK-N-SH cells and blocked apoptosis in cells infected with either gD(-/+) or gD(-/-) virus, whereas glycoprotein B, infected cell protein 22, or the wild-type baculovirus did not block apoptosis. (iii) In SK-N-SH cells, internalized, partially degraded virus particles were detected at 30 min after exposure to gD(-/-) virus but not at later intervals. (iv) Concurrent infection of cells with baculoviruses did not alter the failure of gD(-/-) virus from expressing its genes or, conversely, the expression of viral genes by gD(-/+) virus. These results underscore the capacity of herpes simplex virus to initiate the apoptotic cascade in the absence of de novo protein synthesis and indicate that both gD and gJ independently, and most likely at different stages in the reproductive cycle, play a key role in blocking the apoptotic cascade leading to cell death.

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Figures

FIG. 1
FIG. 1
Determination of PFU equivalents in a stock of gD−/− mutant virus. Stocks of HSV-1(F) and gD−/− were prepared as described in Materials and Methods. Aliquots from these stocks corresponding to the number of cell equivalents indicated were electrophoretically separated in 10% denaturing polyacrylamide gels and electrically transferred onto a nitrocellulose sheet. The samples were reacted with a UL38-specific polyclonal antiserum and with a gD monoclonal antibody. PFU equivalents for gD−/− stocks were estimated from the ratios of UL38 protein. Sizes are indicated in Mr × 1,000.
FIG. 2
FIG. 2
(A to C) Digital images of cells infected with gD−/− and dl20. The cells were fixed at 18 h after infection and reacted with annexin V-FITC. The images were collected with a Zeiss confocal microscope as described in Materials and Methods. (D) Quantification of cells with phosphatidylserine on their surface. Uninfected cells or cells infected with HSV-1(F), gD−/−, or gD−/+ were reacted with annexin V-FITC and analyzed in with a Zeiss confocal microscope. The number of annexin V-positive cells as a function of total number of cells was determined at 8 and 18 h after infection. Each data point reflects the average percentage of annexin V-positive cells present in 10 random fields. The data correspond to the average of two independent experiments.
FIG. 3
FIG. 3
Agarose gels containing electrophoretically separated, ethidium bromide-stained low-molecular-weight DNA from SK-N-SH cultures that were mock infected or infected with gD mutant virus. Subconfluent cultures of SK-N-SH cells were either mock infected or exposed to 10 PFU of HSV-1(F), d120, or gD−/+ or 10 or 100 PFU of gD−/− virus per cell. The cells were harvested at 18 h after infection and processed as described in Materials and Methods.
FIG. 4
FIG. 4
Electron micrographs of thin sections of cells exposed to 100 PFU equivalents of gD−/−. SK-N-SH cells were exposed to 100 PFU equivalents per cell and incubated for 30 min.. They were then harvested, fixed, sectioned, and stained for electron microscopy as described elsewhere (4). The arrow in panel e points to a structure typical of DNA streaming out of damaged capsids.
FIG. 5
FIG. 5
Electrophoretically separated lysates of cells infected with gD−/+ virus or rescuants and reacted with a monoclonal antibody to gI (A) or polyclonal antibody to gJ (B). SK-N-SH cells were mock infected or exposed (10 PFU/cell) to wild-type virus, P6 (gD−/+), or p10 (gD−/+) or individually to three independently derived gD+/+ rescuants. The cells were harvested at 18 h after infection, solubilized, subjected to electrophoresis in denaturing gels, and reacted with a monoclonal antibody to US7 (gI) or polyclonal antibody to US5 (gJ).
FIG. 6
FIG. 6
Electrophoretically separated lysates of cells exposed to baculoviruses carrying the gene encoding gD (A and B), gJ (C), or gB (D) and reacted with the corresponding antibody. SK-N-SH cells were exposed to 10 PFU of HSV-1(F), 10 PFU of recombinant Bac-gD or Bac-gJ, or 18 PFU of Bac-gB per cell. The cells were harvested at 24 h after exposure to HSV-1(F) or recombinant baculovirus (A, C, or D) or at times shown (B), solubilized, subjected to electrophoresis in 10% denaturing polyacrylamide gels, electrically transferred onto a nitrocellulose sheet, and reacted with a polyclonal antibody to gJ or monoclonal antibody to gD or gB.
FIG. 7
FIG. 7
Agarose gels containing electrophoretically separated, ethidium bromide-stained low-molecular-weight DNA from SK-N-SH cell cultures that were mock infected, exposed to wild-type or mutant virus alone, or exposed to both HSV and recombinant baculovirus. (A) Replicate 25-cm2 flasks of subconfluent cultures of SK-N-SH cells were mock infected, exposed to 10 PFU of recombinant baculovirus per cell as indicated or to 100 PFU of Bac-XylE per cell, or doubly infected with 10 PFU of recombinant baculovirus and either 100 PFU of gD−/− or 10 PFU of gD−/+ mutant virus per cell. (B) Replicate cultures of SK-N-SH cells were exposed to 18 PFU of recombinant baculovirus expressing gB per cell or exposed to both 18 PFU of baculovirus and either 100 PFU of gD−/− or 10 PFU of gD−/+ virus per cell. The cells were harvested 24 h after exposure to virus and processed as described in Materials and Methods.
FIG. 8
FIG. 8
Digitized images of SK-N-SH cells exposed to 100 PFU of gD−/− or 10 PFU of gD−/+ virus per cell alone or doubly infected with gD−/− or gD−/+ as described for Fig. 7 and 10 PFU of baculovirus expressing either gD, gJ, or ICP22 per cell. The cells were fixed at 6 h after exposure to the virus and reacted with an antibody specific for ICP0. The images were collected with a Zeiss confocal microscope as described in Materials and Methods.
FIG. 9
FIG. 9
Agarose gels containing electrophoretically separated, ethidium bromide-stained low-molecular-weight DNA from SK-N-SH cultures that were mock infected or infected and either untreated or exposed to TNF-α or anti-Fas antibody. SK-N-SH cells were mock infected or exposed to 10 PFU of HSV-1(F) or 10 PFU of baculovirus expressing gD or gJ per cell. Replicate cultures were exposed to TNF-α (10 μg/ml) at 2 h after infection or to anti-Fas antibody at 6 h after infection. The cells were harvested 18 h after infection and processed as described in Materials and Methods.
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