Ebola virus VP35 antagonizes PKR activity through its C-terminal interferon inhibitory domain

Abstract

Ebola virus VP35 contains a C-terminal cluster of basic amino acids required for double-stranded RNA (dsRNA) binding and inhibition of interferon regulatory factor 3 (IRF3). VP35 also blocks protein kinase R (PKR) activation; however, the responsible domain has remained undefined. Here we show that the IRF inhibitory domain of VP35 mediates the inhibition of PKR and enhances the synthesis of coexpressed proteins. In contrast to dsRNA binding and IRF inhibition, alanine substitutions of at least two basic amino acids are required to abrogate PKR inhibition and enhanced protein expression. Moreover, we show that PKR activation is not only blocked but reversed by Ebola virus infection.

FIG. 1.

VP35 enhances the expression of cotransfected transgenes. (A) C-terminal region of VP35 containing conserved basic amino acids involved in dsRNA binding and interferon inhibition. Substituted amino acids in the mutant VP35_3A are indicated. (B) HEK293 cells were transfected with 50 ng pCMV-GLuc (New England Biolabs), 20 ng pSV40-RLuc (Promega), or 500 ng pRSV-β-Gal along with 500 ng of pcDNA3.1-VP35/Z_NHA (VP35wt) or the corresponding empty vector (noVP35). Twenty-four hours posttransfection, cells were lysed and reporter enzyme activity was determined (n = 6). (C and D) HEK293 cells were transfected with 50 ng pCAGGS-eGFP along with 500 ng of pcDNA3.1-VP35/Z_NHA (VP35wt) or pcDNA3.1-VP35/Z_NHA_3A (VP35_3A) or the corresponding empty vector (noVP35). (C) Twenty-four hours posttransfection, cells were analyzed by fluorescence microscopy. (D) Fluorescence-activated cell sorter analysis was performed with trypsinized cells. The mean fluorescence intensity is indicated in the top right corner of each plot. (E) Western blot analysis was performed with specific primary antibodies against GFP (B-2; Santa Cruz Biotechnology) or β-actin (Abcam) and IRDye800-labeled secondary antibodies (Rockland). Quantitative readout of the stained membrane was performed with the Odyssey infrared imager (LI-COR). The fluorescence intensity of the eGFP signal in relation to the actin background was calculated from three independent experiments. (F) Huh7 cells were transfected with pC-T7/Pol expressing the T7 RNA polymerase, pSV40-RLuc, all components of the EBOV polymerase complex (VP35wt or VP35_3A, NP, VP30, and L), and the 3E-5E-Luc minigenome containing the firefly luciferase gene flanked by the 3′ leader and 5′ trailer sequences of the EBOV genome that regulate viral gene transcription and genome replication. Twenty-four hours posttransfection, firefly luciferase activity was determined and normalized for Renilla luciferase activity. The data from three independent experiments are shown. VP35 expression was determined by Western blot analysis with specific antibodies against the hemagglutinin tag (HA.11; Covance) and β-actin.

FIG. 2.

The IID of VP35 is essential for the PKR antagonistic function. (A) HEK293 cells were transfected as described in the legend to Fig. 1C. Twenty-four hours posttransfection, total RNA was isolated and used for RT-PCR with primers specific for eGFP and GAPDH mRNAs. Reverse transcriptase was omitted to verify the specific amplification of cDNA (lane 1). (B) HEK293 cells were transfected as described in the legend to Fig. 1C. Twenty-four hours posttransfection, cycloheximide (CHX; 100 μg/ml) was added to the cells. Cells were harvested at the indicated time points post-CHX treatment, and eGFP levels were quantified by Western blot analysis as described in the legend to Fig. 1E. The eGFP levels at the different time points and trend lines derived by linear regression in the presence (solid line) or absence (dashed line) of VP35 are shown. The experiment was performed in triplicate. (C) HEK293 cells were transiently transfected with empty vector pcDNA3.1 (EV), exposed to the transfection reagent FuGene HD (FuHD), or left untreated (mock). Twenty-four hours posttreatment, cells were analyzed by Western blot analysis as described above or previously elsewhere (11). (D) HEK293 cells were transfected as described for panel A (left) or with an increased amount of VP35_3A expression vector (2 μg) (right) to compensate for reduced protein levels and analyzed as described for panel C.

FIG. 3.

Substitution of two basic amino acids of the IID is required to abrogate the PKR antagonistic function of VP35. HEK293 cells were transfected as described in legend to Fig. 1C and in addition with VP35 mutants carrying single (A) or double (B) amino acid substitutions to alanine at the indicated positions. The amount of transfected expression plasmid for the double mutants was increased (1 μg) to compensate for reduced expression enhancement. Western blot analysis was performed as described in the legend to Fig. 2.

FIG. 4.

EBOV infection is able to reverse PKR phosphorylation. (A) HEK293 cells were transfected with 50 ng eGFP expression vector or left untreated (mock). (B) Twenty-four hours posttransfection, cells were harvested and subjected to Western blot analysis or infected with EBOV with a multiplicity of infection of 5 where indicated. Twenty-four hours postinfection, cells were analyzed by Western blotting.