The VP35 protein of Ebola virus inhibits the antiviral effect mediated by double-stranded RNA-dependent protein kinase PKR - PubMed (original) (raw)
The VP35 protein of Ebola virus inhibits the antiviral effect mediated by double-stranded RNA-dependent protein kinase PKR
Zongdi Feng et al. J Virol. 2007 Jan.
Abstract
The VP35 protein of Ebola virus is a viral antagonist of interferon. It acts to block virus or double-stranded RNA-mediated activation of interferon regulatory factor 3, a transcription factor that facilitates the expression of interferon and interferon-stimulated genes. In this report, we show that the VP35 protein is also able to inhibit the antiviral response induced by alpha interferon. This depends on the VP35 function that interferes with the pathway regulated by double-stranded RNA-dependent protein kinase PKR. When expressed in a heterologous system, the VP35 protein enhanced viral polypeptide synthesis and growth in Vero cells pretreated with alpha/beta interferon, displaying an interferon-resistant phenotype. In correlation, phosphorylation of PKR and eIF-2alpha was suppressed in cells expressing the VP35 protein. This activity of the VP35 protein was required for efficient viral replication in PKR+/+ but not PKR-/- mouse embryo fibroblasts. Furthermore, VP35 appears to be a RNA binding protein. Notably, a deletion of amino acids 1 to 200, but not R312A substitution in the RNA binding motif, abolished the ability of the VP35 protein to confer viral resistance to interferon. However, the R312A substitution rendered the VP35 protein unable to inhibit the induction of the beta interferon promoter mediated by virus infection. Together, these results show that the VP35 protein targets multiple pathways of the interferon system.
Figures
FIG. 1.
(A) Schematic representation of the genome structure of HSV-1 and the recombinant viruses. The two covalently linked components of HSV DNA, L and S, each consist of unique sequences, UL and US, respectively, flanked by inverted repeats which are designated ab and b'a'. Recombinant virus HSV-BAC contains the wild-type γ134.5 gene. Recombinant virus KY0234 lacks the coding region of the γ134.5 gene (12). Recombinant virus MC0201 was constructed by the HSV-BAC system with the VP35 gene from Ebola virus subtype Zaire replacing both copies of the γ134.5 gene. Recombinant viruses MC0309 and MC0301 are repair viruses in which the VP35 gene of MC0201 was replaced with the wild-type γ134.5 gene and a γ134.5 gene containing an R215L point mutation, respectively. Restriction endonuclease abbreviations: N, NcoI; Be, BstEII; St, StuI; E, EcoRV. (B) Autoradiographic images of viral DNAs. Vero cells were infected with the indicated viruses at 10 PFU per cell. At 18 h after infection, cells were harvested and viral DNAs were prepared for Southern blot analysis as described in Materials and Methods. The VP35 and γ134.5 genes were then detected by hybridization to electrophoretically separated digestion products of viral DNA transferred to a nitrocellulose sheet with either a 32P-labeled EcoRI-XhoI or NotI fragment from the VP35 or γ134.5 gene, respectively. Fragments representing VP35 or γ134.5 are indicated on the right. (C) Expression of the VP35 or γ134.5 gene products. Vero cells were either mock infected or infected with the indicated viruses at 10 PFU per cell. At 18 h postinfection, lysates of cells were prepared and subjected to Western immunoblot analysis with either anti-VP35, anti-γ134.5, or anti-β-actin antibody. The positions of the VP35, γ134.5, and β-actin proteins are shown on the right.
FIG. 2.
(A) Viral response to interferon. Monolayers of Vero cells were not treated (IFN−) or pretreated with human leukocyte alpha interferon (IFN+) (1,000 U/ml; Sigma) for 20 h. Cells were then infected at 0.05 PFU per cell with the indicated viruses. At 72 h postinfection, cells were harvested and freeze-thawed three times, and virus yield was determined on Vero cells. Data represent the averages plus standard deviations (error bars) from three independent experiments. (B) Kinetics of viral growth. Virus infection was carried out as described above for panel A, except that cells were harvested at different time points postinfection. The data represent the averages of duplicate samples, with the standard deviations indicated by the error bars.
FIG. 3.
(A) Viral protein accumulation in the presence and absence of interferon. Monolayers of Vero cells were not treated (−) or were pretreated (+) with human leukocyte alpha interferon (IFN α) (1,000 U/ml; Sigma) for 20 h. Cells were then mock infected or infected with the indicated viruses at 1.0 PFU per cell. At 18 h postinfection, cells were harvested, washed with phosphate-buffered saline, resuspended in disruption buffer, electrophoretically separated on a denaturing 12% polyacrylamide gel, transferred to a nitrocellulose sheet, and probed with a rabbit polyclonal antibody against all HSV-1 antigens as suggested by the manufacturer (Dako Corporation). β-Actin was probed as a loading control. (B) Effect of the VP35 protein on eIF-2α phosphorylation. Vero cells, untreated or pretreated with alpha interferon, were infected with viruses (1 PFU/cell), and lysates of cells were prepared 18 h postinfection. Samples were subjected to immunoblot analysis using rabbit antibody against phospho-Ser51 eIF-2α (Biosource, Inc.) or eIF-2α (Cell Signaling Technology, Inc.). The positions of eIF-2α and phosphorylated eIF-2α (p-eIF2α) are indicated on the left. The ratio between the amounts of phosphorylated eIF-2α and total eIF-2α in each lane were quantitated by densitometry, and the numbers indicate the ratios after normalization to mock-infected cells.
FIG. 4.
Inhibition of PKR activity by the VP35 protein. Vero cells were left untreated (−IFN-α) or pretreated with human alpha interferon (+IFN-α) (1,000 units/ml; Sigma) for 20 h. Cells were then mock infected or infected with the indicated viruses at 1 PFU per cell. At 18 h postinfection, cells were harvested. Cell lysates were prepared and subjected to immunoblotting using rabbit antibody against PKR, phospho-Thr446 PKR (P-PKR), phospho-Ser51eIF-2α (P-eIF2α), or eIF-2α. The ratio between phosphorylated eIF-2α and total eIF-2α in each lane were measured as described in the legend to Fig. 3.
FIG. 5.
(A) Effect of alpha/beta interferon on replication of recombinant viruses in PKR+/+ and PKR−/− mouse fibroblast cells. Cells were pretreated with mouse alpha/beta interferon (1,000 U/ml) for 20 h. Cells were then either mock infected (PKR−) or infected with the recombinant viruses (PKR+) at 0.05 PFU per cell and incubated at 37°C for 48 h. Cells were harvested and freeze-thawed three times, and virus yield was determined on Vero cells. The data represent the averages of triplicate samples, with the standard deviations indicated by error bars.
FIG. 6.
Mutational analysis of the VP35 protein. (A) Expression of VP35 variants. Retroviral vectors expressing GFP, VP35, VP35(R312A), or VP35(dN200) were transfected together with HIVtrans and vesicular stomatitis virus G into 293T cells. Forty-eight hours after transfection, supernatant was used to transduce Vero cells. Expression of VP35 from transduced Vero cells was examined by Western blotting using anti-VP35 and anti-FLAG antibodies. (B) Effects of VP35 variants on viral resistance to interferon. Transduced Vero cells were either treated with 1,000 U/ml human alpha interferon (IFN+) (Sigma) for 20 h or left untreated (IFN−) and infected with a Δγ134.5 mutant at 0.05 PFU per cell. Viruses were harvested at 72 h after infection, and the virus titers on Vero cells were determined. The data represent the averages of three experiments with the standard deviations indicated by error bars. (C) Cytopathic effects. Images were taken from a representative experiment as described above for panel B. (D) Phosphorylation states of PKR and eIF-2α. Transduced Vero cells were either treated with human alpha interferon (+IFNα) (Sigma) or left untreated (−IFNα) and then infected with a Δγ134.5 mutant infection at 1.0 PFU per cell. Infected-cell lysates were prepared at 24 h after infection and subjected to immunoblot analysis using the indicated antibody. The ratio between phosphorylated eIF-2α and total eIF-2α in each lane was measured as described in the legend to Fig. 3. P-PKR, phosphorylated PKR.
FIG. 7.
(A) Effects of VP35 variants on the beta interferon promoter. Plasmids expressing GFP, VP35, VP35(R312A), or dN200 were transfected along with 50 ng beta interferon reporter gene and Renilla luciferase gene under the thymidine kinase promoter. Twenty-four hours after transfection, cells were infected with Sendai virus. The luciferase activities were measured 24 h after infection. The relative beta interferon promoter activities were normalized to Renilla luciferase activity. Data represent the averages plus standard deviations (error bars) from two independent experiments. (B) The expression of VP35 variants in panel A was analyzed by Western blot analysis using antibodies against VP35 (VP35 and R312A) and FLAG (dN200). (C) RNA binding analysis. 293T cells were transfected with plasmid constructs expressing FLAG-tagged VP35, VP35 (R312A), and dN200. Whole-cell lysates (WCL) were prepared and incubated with poly(C), poly(I · C)-conjugated beads (Poly IC), or empty beads. After centrifugation, samples were subjected to electrophoresis and immunoblot analysis with anti-FLAG antibody. As controls for protein expression, aliquots of whole-cell lysates were used to detect the expression of VP35 variants. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gels in panels B and C.
References
- Beattie, E., J. Tartaglia, and E. Paoletti. 1991. Vaccinia virus-encoded eIF-2 alpha homolog abrogates the antiviral effect of interferon. Virology 183:419-422. - PubMed
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