Protein kinase PKR mediates the apoptosis induction and growth restriction phenotypes of C protein-deficient measles virus

Abstract

The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKR(kd) cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (C(ko)) virus but had no effect on the growth of either wild-type (WT) or V knockout (V(ko)) virus. Increased growth of the C(ko) virus in PKR(kd) cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the alpha subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, V(ko), or especially C(ko) virus caused significantly less apoptosis in PKR(kd) cells than in PKR-sufficient cells. Although apoptosis induced by C(ko) virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of C(ko) virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.

FIG. 1.

Growth of V and C mutant viruses compared to WT MV in PKR⁺, PKR^kd, and PKR^kd-con HeLa cells. PKR⁺, PKR^kd, and PKR^kd-con cells were infected with WT, V^ko, or C^ko recombinant virus. (A) Fluorescence images of cells infected at an MOI of 5 were taken at 36 h after infection. UI, uninfected cells. (B) Virus yields from PKR-sufficient and PKR-deficient cells at 48 h after infection at an MOI of 0.1 (low MOI) or 36 h after infection at an MOI of 5 (high MOI) were determined by 50% TCID₅₀ titration on Vero cells. The results shown are the means with standard deviations (n = 3). * , P of <0.05 by Student's t test for comparison of the C^ko virus yields in PKR^kd cells and those in PKR⁺ or PKR^kd-con cells.

FIG. 2.

Viral protein expression levels in WT, V^ko, and C^ko virus-infected PKR⁺, PKR^kd, and PKR^kd-con cells. Cells were infected at an MOI of 0.l, and whole-cell extracts were prepared at 48 h after infection and analyzed by Western immunoblotting by using monospecific antibodies against the indicated MV proteins or β-actin. UI, uninfected cells.

FIG. 3.

PKR and eIF-2α phosphorylation in WT and C^ko virus-infected PKR⁺ cells. (A) Cells were left uninfected (UI) or were infected with WT or C^ko MV at an MOI of 5 TCID₅₀/cell or VVΔE3L at an MOI of 5 PFU/cell. Whole-cell extracts were prepared at the indicated times postinfection. Western blot analyses were performed using antibodies against phospho-PKR (P-PKR), PKR, phospho-eIF-2α (P-eIF-2α), eIF-2α, N, P, V, C, H, GFP, and β-actin. hpi, hours postinfection. (B) Cells were left uninfected or were infected with WT MV at an MOI of 5 TCID₅₀/cell. At 38 h after infection with MV, the cells were superinfected with VVΔE3L at an MOI of 5 PFU/cell where indicated (+). Whole-cell extracts were prepared at the indicated times and analyzed by Western blotting with antibodies against phospho-PKR, phospho-eIF-2α, β-actin, MV C, and VV I3. +, present; −, absent.

FIG. 4.

MV-induced apoptosis is impaired in PKR-deficient cells. PKR⁺, PKR^kd, and PKR^kd-con cells were infected with WT, V^ko, or C^ko virus or left uninfected (UI). (A) Phase-contrast images taken at 36 h postinfection. (B) Results of the colorimetric MTT assay to measure cell viability 48 h postinfection, displayed as percentages of the number of viable uninfected cells (n = 4). * , P of <0.05 by Student's t test for comparison of the C^ko virus yields in PKR^kd cells and those in PKR⁺ or PKR^kd-con cells. (C) Western immunoblot analyses performed on whole-cell extracts prepared at 36 h postinfection, using antibodies against human PARP and β-actin. The quantity of PARP cleavage based on the immunoblots, expressed as a ratio of PARP85 to total PARP [PARP85 ÷ (PARP85 + PARP116)], is shown below each lane.

FIG. 5.

Inhibition of apoptosis does not restore the WT host range phenotype in C^ko MV. PKR^kd and PKR^kd-con cells infected with C^ko or WT virus (MOI of 5) or left uninfected (UI) were treated with medium alone or medium containing the caspase inhibitor z-VAD-fmk (100 μM). Cells were analyzed at 36 h postinfection. (A) Phase-contrast images. (B) Western immunoblot analyses using antibodies against human PARP, GFP, and β-actin. (C) Fluorescence images. (D) Virus yields determined by 50% TCID₅₀ titration on Vero cells. The results shown are means ± standard deviations (n = 4).