PKR acts early in infection to suppress Semliki Forest virus production and strongly enhances the type I interferon response

Abstract

The double-stranded RNA-activated protein kinase (PKR) is a key regulator of protein translation, interferon (IFN) expression and cell survival. Upon infection of vertebrate cells in continuous culture, the alphavirus Semliki Forest virus (SFV) initiates apoptosis and IFN synthesis. To determine the effect of PKR on SFV infection, we studied the course of infection in wild-type (wt) mice, mice with a genetic deletion of PKR (PKR-/-) and mouse embryo fibroblasts (MEFs) derived from these mice. In MEFs, PKR delayed virus protein synthesis, production of infectious virus and caspase-3-activated cell death and reduced the yield of infectious virus by 90%. Small interfering RNA suppression of PKR levels in NIH-3T3 cells also reduced virus production and apoptosis. In MEFs, PKR was not required for initiation of IFN-beta gene transcription, but contributed strongly to the magnitude of this response. Levels of IFN-beta transcripts in PKR-/- MEFs at 8 h were 80% lower than those in wt MEFs and levels of functional IFN at 24 h were 95% lower. Following infection of wt and PKR-/- mice, SFV4 and SFV A7(74) were avirulent. PKR increased levels of serum IFN and the rate of clearance of infectious virus from the brain. In summary, in response to SFV, PKR exerts an early antiviral effect that delays virus protein production and release of infectious virus and, whilst PKR is not required for induction of apoptosis or activation of the type I IFN response, it strongly augments the type I IFN response and contributes to clearance of infectious virus from the mouse brain.

Fig. 1.

Virus RNA copy number (μg total RNA)⁻¹ from wt (□) and PKR^−/− (▴) MEFs infected (m.o.i. of 50) with SFV4, as measured by QPCR. Each point is the mean of replicate samples from three parallel cultures. Error bars indicate sd.

Fig. 2.

Metabolic labelling of newly synthesized proteins in SFV4-infected (m.o.i. of 50) PKR^−/− (a) or wt (b) MEFs. After labelling with [³⁵S]Met/Cys, cells were lysed at the indicated time points and equal amounts of protein (μg) were run on 12 % polyacrylamide/SDS gels.

Fig. 3.

Production of SFV4 virus from wt (□) and PKR^−/− (▴) MEFs following infection at an m.o.i. of 50 (a) and an m.o.i. of 1 (b). Each point is the mean of replicate samples from three parallel cultures. Error bars indicate sd.

Fig. 4.

(a) Viability (WST-1) of wt (□) and PKR^−/− (▴) MEFs following infection (m.o.i. of 50) with SFV4. Each point represents the mean of four samples. Error bars indicate sd. *P<0.05 (Mann–Whitney test). (b) Levels of activated caspase-3 (arbitrary units), as determined by caspase-3 activation assay. wt (□) and PKR^−/− (▴) MEFs were infected with SFV4 (m.o.i. of 50). Cells were lysed and equal amounts (μg) were assayed for caspase-3 activation. Each point represents the mean of three samples. Error bars indicate sd.

Fig. 5.

(a) Virus titres in the supernatant and (b) viability of replicate cultures of NIH-3T3 cells pre-treated with control siRNA (empty bars) or PKR siRNA (filled bars) and infected (24 h later) with SFV4 (m.o.i. of 1.0 or 50). Each value is the mean of replicate samples from three parallel cultures. Error bars indicate sd. *P<0.05; **P<0.01 (Mann–Whitney test). The experiment was repeated twice (at each m.o.i.) with similar results.

Fig. 6.

wt (empty bars) and PKR^−/− (filled bars) MEFs were grown to confluence and infected (m.o.i. of 10) with SFV4 or mock-infected with PBS. At 0, 2, 3, 4, 8 and 12 h post-infection, cells were harvested and RNA was extracted and reverse-transcribed into cDNA to determine levels of (a) IFN-β transcripts, (b) IFN-α transcripts and (c) β-actin transcripts by QPCR. Each bar represents the mean of two repeat experiments (each done with different preparations of MEFs), each of which included three replicate cultures each assayed in triplicate. The limit of detection is at the intersection of the axes. Error bars indicate sem. *P<0.05 (Mann–Whitney test).

Fig. 7.

wt (empty bars) and PKR^−/− (filled bars) MEFs were grown to confluence and infected (m.o.i. of 10) with SFV4 or mock-infected with PBS. Supernatants sampled from MEFs at 18 and 24 h post-infection were assayed for functional IFN by CPERA. Each bar represents the mean of two repeat experiments (each done with different preparations of MEFs), each of which included four cultures assayed in triplicate. Error bars indicate sem. *P<0.05 (Mann–Whitney test) between wt and PKR^−/− cells.

Fig. 8.

Groups (n=25) of 4–5-week-old wt and PKR^−/− mice were infected ip with 5000 p.f.u. SFV A7(74). At various times post-infection, brain virus titres were determined by plaque assay. Each bar represents the mean (n=6) virus titre from wt (empty bars) or PKR^−/− (filled bars) mouse brains. Error bars indicate sd. The limit of detection of the assay was 10^2.4 p.f.u. (g brain)⁻¹. *P<0.05 (Mann–Whitney test).