A five-amino-acid deletion of the eastern equine encephalitis virus capsid protein attenuates replication in mammalian systems but not in mosquito cells

Abstract

Eastern equine encephalitis virus (EEEV) is a human and veterinary pathogen that causes sporadic cases of fatal neurological disease. We previously demonstrated that the capsid protein of EEEV is a potent inhibitor of host cell gene expression and that this function maps to the amino terminus of the protein. We now identify amino acids 55 to 75, within the N terminus of the capsid, as critical for the inhibition of host cell gene expression. An analysis of stable EEEV replicons expressing mutant capsid proteins corroborated these mapping data. When deletions of 5 to 20 amino acids within this region of the capsid were introduced into infectious EEEV, the mutants exhibited delayed replication in Vero cells. However, the replication of the 5-amino-acid deletion mutant in C710 mosquito cells was not affected, suggesting that virus replication and assembly were affected in a cell-specific manner. Both 5- and 20-amino-acid deletion mutant viruses exhibited increased sensitivity to interferon (IFN) in cell culture and impaired replication and complete attenuation in mice. In summary, we have identified a region within the capsid protein of EEEV that contributes to the inhibition of host gene expression and to the protection of EEEV from the antiviral effects of IFNs. This region is also critical for EEEV pathogenesis.

FIG. 1.

Amino acids 55 to 75 of the EEEV capsid protein are necessary for the inhibition of gene expression. (A) Schematic representation of the EEEV capsid deletion mutant proteins generated in this study. An HA tag was introduced at the N terminus of the sequence corresponding to the coding region to confirm expression. (B) 293T cells were cotransfected with a GFP reporter plasmid and either an empty vector or a plasmid expressing either the full-length capsid or one of the capsid mutant proteins. Twenty-four hours posttransfection, the cells were examined for GFP expression by microscopy. (C) An experiment was performed as described in the legend to panel B, except that a reporter plasmid expressing firefly luciferase protein was used and cells were harvested and assayed for luciferase activity. The black and white bars and the error bars represent the means ± the standard errors for samples from three separately transfected wells. The experiment was repeated at least twice with consistent results. (D) Western blotting was performed to detect the expression of the full-length capsid and capsid deletion mutant proteins (by detecting the HA tag). The expression of α-tubulin was also measured as a loading control.

FIG. 2.

The expression of amino acids 55 to 75 of the EEEV capsid is sufficient to inhibit gene expression. (A) 293T cells were transfected with an empty vector or a plasmid encoding the full-length capsid, the capsid N terminus (N-ter), the capsid C terminus (C-ter), or one of the capsid deletion mutant proteins. One day after transfection, cells were treated with IFN, and 12 h after treatment, cell lysates were obtained and analyzed by Western blotting for the expression of STAT-1 and GAPDH as a loading control. (B) The quantification of STAT-1 expression was performed using a phosphorimager. The STAT-1 expression level in the vector-transfected cells was set as 100%. (C) 293T cells were cotransfected with a firefly luciferase expression plasmid and a plasmid encoding either GFP fused to a simian virus 40 NLS (S-GFP), the full-length capsid fused to GFP, the region of amino acids 55 to 75 of the capsid fused to GFP (GFP-55-75), or the N terminus of the capsid fused to GFP. Twenty-four hours posttransfection, the cells were lysed and assayed for luciferase activity. The bars and error bars represent the means ± the standard errors for samples from three separately transfected wells. The expression of the constructs was confirmed using anti-GFP antibodies and tubulin as a loading control.

FIG. 3.

An EEEV replicon expressing a capsid with the region of amino acids 55 to 75 deleted is not cytotoxic. (A) Schematic representation of the EEEV replicons used in this study. EEE rep, EEEV replicon lacking the capsid gene; EEE rep capsid, EEEV replicon encoding the capsid; EEE rep cap d55-75, EEEV replicon with the deletion of the sequence encoding amino acids 55 to 75 within the capsid gene; nsP1 to nsP4, genes for nonstructural proteins 1 to 4; Luc, luciferase gene; PAC, puromycin acetyltransferase gene (encoding puromycin resistance); C, capsid gene; arrows labeled 26S, 26S subgenomic promoters. (B) Equal amounts of in vitro-transcribed replicon RNAs were introduced into BHK cells by electroporation, and 24 h after electroporation, equal numbers of cells were lysed and assayed for luciferase activity.

FIG. 4.

Capsid mutant viruses replicate less efficiently than parental EEEV in Vero cells, but Δ65-69 is not impaired in mosquito cells. Vero cells and C710 cells were infected with parental FL93-939 or the Δ55-75 or Δ65-70 capsid deletion mutant, and at the indicated time points, supernatants were harvested and virus titers were determined by plaque assays (n = 3). The error bars indicate standard errors of the means.

FIG. 5.

Subcellular localization of EEEV capsid proteins from wild-type and mutant viruses in Vero cells. Vero cells were infected with parental and capsid mutant EEEVs, and confocal microscopy studies were performed using anti-EEEV capsid-specific antibodies. The capsid proteins from strain FL93-939 (top row) and mutants Δ55-75 (middle row) and Δ65-69 (bottom row) were concentrated in the nuclei 8 hpi.

FIG. 6.

Parental and capsid mutant EEEVs induce the phosphorylation of eIF-2α. Vero cells were mock infected or infected with EEEV strain FL93-939 or the Δ55-75 or Δ65-69 capsid mutant virus (MOI = 0.1), and at 24 hpi, cells were lysed and Western blotting was performed to detect the phosphorylated eIF-2α (phospho-eIF2 alpha), total eIF-2α, and α-tubulin as a loading control. Similar results were observed at 12 hpi (data not shown).

FIG. 7.

Capsid mutant viruses are completely attenuated in mice. Six-week-old mice were infected subcutaneously with 1,000 PFU of wild-type parental FL93-939 virus or Δ55-75 or Δ65-70 capsid mutant virus. (A) Viremia generated in the animals as measured by plaque assays (n = 3) at the indicated days postinfection. Asterisks indicate that the titer was below the limit of detection (2 log₁₀ PFU/ml). (B) Survival rates (n = 6).