Overexpression of promyelocytic leukemia protein precludes the dispersal of ND10 structures and has no effect on accumulation of infectious herpes simplex virus 1 or its proteins

Abstract

A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing the M(r) 69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of alpha, beta, or gamma groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the alpha 0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication.

FIG. 1.

PML cloned in a plasmid or baculovirus is expressed and modified by SUMO-I. (A and B) HEp-2 cells were transfected with pAcCMV (lanes 4 and 8) or pAcCMV-PML (lanes 1, 2, 5, and 6) or were untransfected (lanes 3 and 7). After 48 h of incubation the cells were harvested, solubilized, subjected to electrophoresis in denaturing gels, and reacted with a mouse monoclonal antibody to PML (A) or with a rabbit polyclonal antibody to SUMO-I (B). (C) Lysates of cells infected with two different preparations of Bac-PML (lanes 9 and 10) were solubilized, subjected to electrophoresis in denaturing gels, and reacted with a mouse monoclonal antibody to PML. Because of the large amount of PML expressed in these cells, the exposure to the antibody was very short, too short to detect endogenous PML in panel A, lanes 3 and 4.

FIG. 2.

Overexpression of PML results in the formation of large nuclear structures (ND10) containing CBP and Daxx. pHF fibroblast slide cultures were infected with Bac-PML (d to f and j to l) or wild-type baculovirus (a to c and g to i) as described in Materials and Methods. The cells were fixed and reacted with a mouse monoclonal antibody to PML (b, c, e, f, h, i, k, and l) or a rabbit polyclonal antibody to CBP (a, c, d, and f) or Daxx (g, i, j, and l). The secondary antibodies were anti-mouse immunoglobulin G (IgG) conjugated to fluorescein isothiocyanate and an anti-rabbit IgG conjugated to Texas red. Left and middle columns, single-color images captured separately; right column, merged images. The yellow visualized in the overlaid images represents colocalization of PML and CBP or PML and Daxx. The images were captured with a Zeiss camera (AxioCam) and software provided by Zeiss. The digitized images were not modified subsequent to capture.

FIG. 3.

Overexpressed PML remains associated with enlarged ND10 structures but does not affect the localization of ICP0 in the course of infection of pHF fibroblasts. pHF fibroblast slide cultures were infected with Bac-PML (d to f, j to l, p to r, and v to x) or wild-type baculovirus (a to c, g to i, m to o, and s to u) and then either mock infected (a to f) or infected with HSV-1(F) (g to x) as described in Materials and Methods. The cells were fixed at the indicated times and reacted with a mouse monoclonal antibody to PML and a rabbit polyclonal antibody to ICP0 as indicated at the top of each column. The secondary antibodies were anti-rabbit immunoglobulin G (IgG) conjugated to Texas red and an anti-mouse IgG conjugated to FITC. The images were captured as described in the legend to Fig. 2 and were not modified subsequent to capture.

FIG. 4.

ICP0 colocalizes, but does not overlap, with PML. pHF fibroblast slide cultures were infected with Bac-PML and HSV-1(F) at 3 h after HSV-1 infection and were treated as indicated in the legend for Fig. 2. The images were captured with a Zeiss confocal microscope with the aid of software provided by the manufacturer and were not modified subsequent to capture.

FIG. 5.

CBP and Daxx colocalize with PML in enlarged ND10 structures throughout the replicative cycle of HSV-1. pHF fibroblast slide cultures were exposed to Bac-PML (d to f, j to l, p to r, and v to x) or wild-type baculovirus (a to c, g to i, m to o, and s to u). The cultures were then mock infected or infected with HSV-1(F) as indicated. At 12 h after mock infection or infection with HSV-1(F) the cells were fixed and reacted with the antibody to CBP (conjugated to Texas red), gD (conjugated to FITC), or Daxx (conjugated to Texas red) as shown at the top. The procedures were as described in Materials and Methods. Single-color images captured separately (first, second, fourth and fifth columns from the left) and merged images (third and sixth columns) are shown. The images were captured with a Zeiss confocal microscope with the aid of software provided by the manufacturer and were not modified subsequent to capture.

FIG. 6.

In cells overexpressing PML, sumoylated proteins decrease with time but are detectable late in infection. Replicate cultures of pHF fibroblasts were exposed to wild-type baculovirus (PML −) or baculovirus expressing PML. After 10 h of incubation, the cells were mock infected or exposed to 5 PFU of HSV-1(F) (A) or 1 PFU of the R7910 (α0) mutant (B) per cell. The cultures were harvested at 2, 4, 8, or 12 h after infection, solubilized, and subjected to electrophoresis in denaturing polyacrylamide gels, and the products were transferred to a nitrocellulose sheet and reacted with a monoclonal antibody to PML (left) or a rabbit polyclonal antibody to SUMO-I (right).

FIG.7.

Electron photomicrographs of enlarged ND10 structures overexpressing PML and infected with HSV-1(F). pHF fibroblasts were harvested 3 h after infection with HSV-1(F) and processed for immunoelectron microscopy as described in Materials and Methods. Thin sections of cells were labeled with a mouse monoclonal antibody to PML (Santa Cruz Biotechnology) and then reacted with a goat anti-mouse immunoglobulin G (IgG) antibody conjugated with 15-nm colloidal gold (GMHL-15) as a secondary antibody (Ted Pella). (A) Low magnification of a cell showing multiple ND10 structures delineated by circles and containing multiple 15-nm gold particles. (B) Higher magnification showing individual ND10 structures delineated by the circles and containing 15-nm gold particles indicating the presence of PML. (C) Thin sections of cells were labeled simultaneously with a mouse monoclonal antibody to PML and a rabbit antibody to ICP0 and then reacted with a goat anti-mouse IgG antibody conjugated with 15-nm colloidal gold as a secondary antibody and a goat anti-rabbit IgG antibody conjugated with 5-nm colloidal gold (GFAR-5). This high magnification of a single ND10 structure shows the presence of both ICP0 (5-nm) and PML (15-nm) particles.

FIG. 8.

Overexpression of PML does not affect the expression of viral proteins. Replicate cultures of pHF fibroblasts were exposed to wild-type baculovirus (PML −) or baculovirus expressing PML (PML +). At 10 h later, the cells were mock infected or exposed to 5 PFU of HSV-1(F) (A) or 1 PFU of the R7910 (α0-null) mutant (B) per cell. The cells were harvested at 2, 4, 8, or 10 h after the 2-h adsorption interval, solubilized, subjected to electrophoresis in denaturing gels, and reacted with antibodies as described in Materials and Methods. (C) Same procedures as for panels A and B with HEp-2 cells.