Capsid protein of eastern equine encephalitis virus inhibits host cell gene expression

Abstract

Eastern equine encephalitis virus (EEEV) causes sporadic but often severe cases of human and equine neurological disease in North America. To determine how EEEV may evade innate immune responses, we screened individual EEEV proteins for the ability to rescue the growth of a Newcastle disease virus expressing green fluorescent protein (NDV-GFP) from the antiviral effects of interferon (IFN). Only expression of the EEEV capsid facilitated NDV-GFP replication. Inhibition of the antiviral effects of IFN by the capsid appears to occur through a general inhibition of cellular gene expression. For example, the capsid inhibited the expression of several reporter genes under the control of RNA polymerase II promoters. In contrast, capsid did not inhibit expression from a T7 RNA polymerase promoter construct, suggesting that the inhibition of gene expression is specific and is not a simple manifestation of toxicity. The inhibition correlated both with capsid-induced phosphorylation of eukaryotic initiation factor 2 alpha and with capsid-mediated inhibition of cellular mRNA accumulation. Mapping analysis identified the N terminus as the region important for the inhibition of host gene expression, suggesting that this inhibition is independent of capsid protease activity. Finally, when cell lines containing EEEV replicons encoding capsid were selected, replicons consistently acquired mutations that deleted all or part of the capsid, for example, amino acids 18 to 135. Given that the amino terminus of the capsid is required to inhibit host cell gene expression, these data suggest that capsid expression from the replicons is ultimately toxic to host cells, presumably because of its ability to inhibit gene expression.

FIG. 1.

Expression of the EEEV capsid protein counteracts the antiviral effects of the IFN response. A549 cells were transfected with empty plasmid or a plasmid encoding GFP (as a control for transfection efficiency), Ebola virus VP35, Nipah virus W, or the indicated EEEV proteins, and 1 day later, cells were infected with NDV-GFP. Only plasmids expressing Ebola virus VP35, Nipah virus W, and EEEV capsid rescued NDV-GFP replication. The results shown are representative of at least three different experiments. The NDV-GFP panel shows replication of this virus in untransfected cells.

FIG. 2.

EEEV capsid is a general inhibitor of gene expression. (A and B) Vero cells were cotransfected with the ISG54 promoter-CAT reporter plasmid, a constitutively expressed luciferase reporter plasmid, and empty vector or a plasmid expressing EEEV capsid or Nipah virus V protein. At 24 hours posttransfection, the cells were mock treated or treated with 1,000 U human IFN-β/ml for 24 h. Cells were harvested and then assayed for CAT and luciferase activities. (A) Nonnormalized CAT activity, with the untreated empty vector control value set to 1. (B) Relative CAT activity upon normalization to the luciferase control. (C and D) 293T cells were cotransfected with the IFN-β promoter-CAT reporter plasmid, a constitutively expressed luciferase reporter plasmid, and empty vector or a plasmid expressing EEEV nsP2, EEEV capsid, Ebola virus VP35, or Nipah virus W. At 24 hours posttransfection, the cells were mock infected or infected with Sendai virus (SeV) for 24 h. Cells were harvested and then assayed for CAT and luciferase activities. (C) Nonnormalized CAT activity, with the untreated empty vector control value set to 1. (D) CAT activity upon normalization to the luciferase control. The results are representative examples from a set of at least three separate experiments. (E) 293T cells were cotransfected with a luciferase reporter plasmid and either empty vector or a plasmid expressing EEEV capsid. At 24 h posttransfection, the cells were harvested and assayed for luciferase activity. The data represent the means ± standard errors among samples from three separately transfected wells. The experiment was repeated at least twice, with consistent results. (F) An experiment was performed as described for panel E, except that a plasmid expressing GFP was used and cells were examined by microscopy for GFP expression. The experiment was repeated at least twice, with consistent results.

FIG. 3.

Expression of EEEV capsid inhibits RNA polymerase II-directed gene expression. BSRT7 cells were cotransfected with a firefly luciferase reporter plasmid expressed from an RNA polymerase II promoter (in the plasmid pCAGGS) (52) or from a T7 promoter and with increasing concentrations of a plasmid expressing EEEV capsid. At 24 h posttransfection, the cells were harvested and assayed for luciferase activity. Average relative luciferase activities are reported, and the no-capsid control value was set to 100%. The error bars represent standard errors among samples from three separately transfected wells. The experiment was repeated at least twice, with consistent results.

FIG. 4.

Expression of EEEV capsid protein induces phosphorylation of the α subunit of eIF2. (A) 293T cells were cotransfected with a luciferase reporter plasmid and empty vector (vector) or a plasmid expressing EEEV capsid, EEEV structural proteins, or the LCV NSs protein. Twelve or 24 h after transfection, the cells were harvested, and Western blotting was performed to detect the phosphorylation of eIF2α, total eIF2α, and α-tubulin as a loading control. Reductions in luciferase activity were observed for cells transfected with the capsid, the EEEV structural proteins, and LCV NSs (data not shown). (B) 293T cells were mock infected or infected with EEEV strain FL93-939 (MOI = 3), and at 5 and 24 h p.i., cells were lysed and Western blotting was performed to detect the phospho-eIF2α, total eIF2-α, and α-tubulin as a loading control.

FIG. 5.

Expression of EEEV capsid inhibits host mRNA accumulation. 293T cells were cotransfected with a plasmid expressing the influenza A/PR8/34 (H1N1) virus NS1 protein and either empty vector or increasing amounts of a plasmid expressing EEEV capsid. At 24 h posttransfection, RNAs were extracted from the cells for quantitative real-time RT-PCR (A), and cell lysates were used for Western blot analyses of NS1, HA capsid, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (B). The data represent means ± standard errors among samples from two independently transfected wells. The experiment was repeated twice and gave consistent results.

FIG. 6.

The N terminus of the EEEV capsid mediates the inhibition of gene expression. (A) 293T cells were cotransfected with a plasmid expressing the GFP reporter and either empty vector or a plasmid expressing the N terminus (aa 1 to 126) or the C terminus (aa 127 to 261) of the EEEV capsid. At 24 h posttransfection, cells were examined by microscopy for GFP expression. (B) The experiment was performed as described for panel A, except that a luciferase reporter plasmid was used and the cells were harvested and assayed for luciferase activity. (C) 293T cells were cotransfected with a luciferase reporter plasmid and empty vector (vector) or a plasmid expressing EEEV full-length capsid or the N terminus (aa 1 to 126) or C terminus (aa 127 to 261) of the capsid. Twenty-four hours after transfection, the cells were harvested and Western blotting was performed to detect the phosphorylation of eIF2α, total eIF2α, and α-tubulin as a loading control. (D) 293T cells were transfected with empty plasmid or with a plasmid encoding the full-length capsid or the N-terminal or C-terminal half of the capsid. At 24 h posttransfection, the cells were treated with human IFN-β, and 12 h after treatment, RNAs were extracted from the cells for quantitative real-time RT-PCR of STAT-1 and ISG54 mRNAs. The data represent the means ± standard errors among samples from three wells in two experiments.

FIG. 7.

The capsid gene is mutated in stably selected EEEV replicons. (A) Schematic representation of the EEEV replicons constructed for this study. nsP1 to nsP4, genes for nonstructural proteins 1 to 4; Luc, luciferase gene; PAC, puromycin acetyltransferase gene (encoding puromycin resistance); C, capsid gene; arrows with 26S, 26S subgenomic promoters. (B) BHK cells were electroporated with equal amounts of in vitro-transcribed replicon RNA, and 12 h after electroporation, puromycin selection was applied to the cells. Surviving cells were expanded, and RNAs were extracted from individual clones for RT-PCR amplification and sequence analysis. The figure shows the deletions found in the capsid and luciferase genes in the stable cell lines containing the EEEV replicons with the capsid gene. EEE rep capsid, EEEV replicon encoding capsid; EEE rep, EEEV replicon lacking capsid. The gray areas indicate the deleted regions in the stable capsid replicon cell lines.