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Herpes simplex virus type 1 immediate-early protein vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase - PubMed

Herpes simplex virus type 1 immediate-early protein vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase

J Parkinson et al. J Virol. 1999 Jan.

Abstract

Herpes simplex virus type 1 (HSV-1) infection causes the active degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and this process is reliant on the expression of the HSV-1 immediate-early protein Vmw110. In this study we investigated in more detail the mechanism by which the degradation occurs, the domains of Vmw110 which are required, and whether Vmw110 is by itself sufficient for the effect. We found that proteasome inhibitors prevented the degradation of DNA-PKcs, indicating the involvement of a proteasome pathway. Furthermore, the continued activity of DNA-PK during infection in the presence of these inhibitors indicated that Vmw110 does not directly alter the enzyme activity of DNA-PKcs prior to its degradation in a normal infection. Indeed, Vmw110 was found to bind to neither the catalytic nor Ku subunits of DNA-PK. Using mutant Vmw110 viruses we show that the RING finger domain of Vmw110 is essential for the induced degradation of DNA-PKcs but that the ability of Vmw110 to bind to a cellular ubiquitin-specific protease (HAUSP) is not required. When expressed in the absence of other viral proteins, Vmw110 was sufficient to cause the degradation of DNA-PKcs, indicating that the effect on the stability of DNA-PKcs was a direct consequence of Vmw110 activity and not an indirect Vmw110-dependent effect of virus infection. Finally, the Vmw110-induced degradation of DNA-PKcs and loss in DNA-PK activity appears to be beneficial to HSV-1 infection, as virus replication was more efficient in cells lacking DNA-PKcs, especially at low multiplicities of infection.

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Figures

FIG. 1
FIG. 1

Effect of Vmw110 mutant viruses on DNA-PKcs protein levels. HeLa S3 cells were mock infected or infected with wild-type (WT) 17syn+ virus and Vmw110 mutant viruses as shown at 10 PFU per cell. At 24 hpi cells were harvested into Laemli buffer and samples were analyzed by Western blotting. (A) Blot probed with polyclonal anti-DNA-PKcs DPK1 at a dilution of 1/3,000; (B) same blot reprobed with MAb anti-Vmw175 10176 at a dilution of 1/2,000.

FIG. 2
FIG. 2

Prevention of virus-induced DNA-PKcs degradation by the proteasome inhibitor MG132. HeLa S3 cells were mock infected or infected with the viruses as shown at 10 PFU per cell in the presence (+) or absence (−) of 10 μM MG132. Samples were harvested 24 hpi and analyzed by Western blotting. (A) Blot probed with polyclonal anti-DNA-PKcs DPK1 at a dilution of 1/3,000; (B) same blot reprobed with MAb anti-Vmw175 10176 at a dilution of 1/2,000. WT, wild type.

FIG. 3
FIG. 3

DNA-PKcs does not coimmunoprecipitate with Vmw110. HeLa S3 cells were mock infected or infected with wild-type (WT) virus at 10 PFU per cell, and extracts for immunoprecipitation with MAb anti-Vmw110 11060 were made 8 hpi. Proteins that were in the total cell extract (lanes T), that were bound to the preclear beads (lanes P), that immunoprecipitated (lanes B), and that remained in the supernatant after precipitation (lanes S) were analyzed by Western blotting. Blots were probed sequentially with polyclonal anti-DNA-PK DPK1 at a dilution of 1/3,000 (A), MAb anti-Vmw110 11060 at a dilution of 1/10,000 (B), and polyclonal anti-HAUSP r201 at a dilution of 1/1,000 (C). The arrowhead in panel C indicates the band corresponding to HAUSP.

FIG. 4
FIG. 4

Virus-induced degradation of DNA-PKcs seen by confocal microscopy. HeLa S3 cells were mock infected (A) or infected with wild-type syn17+ (B and C) or FXE (D and E) viruses at 10 PFU per cell. The cells were fixed at 8 hpi and costained with MAb anti-DNA-PKcs 18-2 at a dilution of 1/75 (A, B, and D) and polyclonal anti-Vmw110 r190 at a dilution of 1/1,000 (C and E). The bar indicates 5 μm.

FIG. 5
FIG. 5

Vmw110 expressed from a plasmid is sufficient to induce DNA-PKcs degradation. HeLa S3 cells were transfected with plasmid pCI110 expressing Vmw110, processed for immunofluorescence after 24 h, and costained with polyclonal anti-DNA-PKcs DPK1 at a dilution of 1/75 and MAb anti-Vmw110 11060 at a dilution of 1/2,000. Arrows indicate transfected cells. Note that the upper right cell only weakly expressed Vmw110 but that, despite this, DNA-PKcs was still eliminated.

FIG. 6
FIG. 6

Replication of wild-type (WT) and dl1403 viruses in MO59K and MO59J cells. MO59K and MO59J cells were infected with wild-type or dl1403 virus at the MOI shown, and virus yields were titrated on BHK cells 2 days pi. Graphs show virus yields (log titers) versus input MOI (PFU per cell) for wild-type and dl1403 viruses in MO59K (gray bars) and MO59J (black bars) cells from two separate experiments. (Note that as the comparison is between the two cell lines and not between the two viruses, the scale range for log titers is different for each virus. In experiment 2 the effect of the MOI was greater on MO59K cells than in experiment 1, so the lower limit of the graph was reduced.)

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