Semliki Forest virus nonstructural protein 2 is involved in suppression of the type I interferon response

Abstract

The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist.

FIG. 1.

(A) BHK (left panel) or wt B6 MEF (right panel) cells were infected with SFV4 or SFV4-RDR at a MOI of 0.05, samples were taken at the indicated time points, and infectious virus was quantified by plaque assay on BHK cells. Data points are averages for three experiments. (B) sv129 IFNAR1^−/− (left panel) or wt sv129 (right panel) MEFs were infected with SFV4 or SFV4-RDR at a MOI of 0.05, samples were taken at the indicated time points, and infectious virus was quantified by plaque assay on BHK cells. Data points are averages for two experiments. (C) Comparison of viral plaque sizes of SFV4 and SFV4-RDR on BHK cells, wt B6 MEFs, sv129 IFNAR1^−/− MEFS, and wt sv129 MEFs.

FIG. 2.

(A) Total cellular RNA was taken at 12 hpi from MEFs infected with SFV4 or SFV4-RDR and analyzed by QPCR for IFN-β mRNA levels. Bars represent the mean of triplicate measurements from a representative experiment, error bars are standard deviations, and the asterisk indicates a P value of <0.05 (Student's t test). (B) Supernatants from SFV4- or SFV4-RDR-infected MEFs were taken at 24 hpi and analyzed by bioassay for levels of functional IFN. Bars represent the mean of triplicate measurements from a representative experiment, error bars are standard deviations, and the asterisk indicates a P value of <0.05 (Student's t test). (C) Supernatants from infected MEFs were taken at 24 hpi and analyzed by ELISA for levels of TNF-α. Bars represent the mean of triplicate measurements from one representative experiment of three.

FIG. 3.

(A) Total cellular RNA was taken at 12 hpi from mock-infected MEFs (black bars) or MEFs infected with SFV4 (gray bars) or SFV4-RDR (white bars) and analyzed by QPCR for β-actin (left panel) and GAPDH (right panel) mRNA levels. Each bar represents the mean of triplicate measurements from a representative experiment, and error bars are standard deviations. (B) Total cellular and viral RNA synthesis was visualized by [¹⁴C]uridine labeling between 1 and 3 hpi (lanes 1 to 3), 3 and 5 hpi (lanes 4 to 6), or 5 and 7 hpi (lanes 7 to 9) in wt B6 MEFs mock infected (lanes 1, 4, and 7), infected at an MOI of 50 with SFV4 (lanes 2, 5, and 8), or infected at a MOI of 50 with SFV4-RDR (lanes 3, 6, and 9). Positions of the viral genomic 42S and subgenomic 26S RNAs are indicated on the right. (C) Densitometry analysis was performed on the intensities of cellular RNA bands labeled with an asterisk (left panel) or two asterisks (right panel) in panel B between 2 and 5 hpi. Data are averages from four experiments, and error bars are standard deviations.

FIG. 4.

(A) The kinetics of protein synthesis at early times postinfection in SFV4-infected (upper panel) or SFV4-RDR-infected (lower panel) MEFs (each at a MOI of 10) was analyzed by [³⁵S]methionine labeling. Positions of cellular actin and viral p62, E1, and capsid proteins are indicated on the right. (B) Densitometry was performed on the intensity of the band corresponding to actin. Data are averages from three experiments, and error bars are standard deviations. (C) The phosphorylation state of eIF2α in these cells 5 h postinfection or mock infection (M) was determined by immunoblotting for phospho-eIF2α (upper panel) and related to total eIF2α by stripping and reprobing the membrane (lower panel).

FIG. 5.

B6 MEFs were infected at a MOI of 1 (A) or 50 (B) with SFV4 or SFV4-RDR or were mock infected. Cells were fixed at 5 hpi (A) or 3 hpi (B) and immunostained with antibodies to SFV E2 or replicase (green fluorescence) and either IRF-3 (upper panels, red fluorescence) or NF-κB p65 (lower panels, red fluorescence). Bar, 50 μm.