ISG56/IFIT1 is primarily responsible for interferon-induced changes to patterns of parainfluenza virus type 5 transcription and protein synthesis

Abstract

Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2'-O-methyltransferase activity, an enzyme that methylates the 2'-hydroxyl group of ribose sugars in the 5'-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2'-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5.

Fig. 1.

Transient expression of ISG56/IFIT1 inhibits PIV5. Hep2 cells were transiently transfected with a plasmid containing haemagglutinin (HA)-tagged ISG56/IFIT1. At 24 h post-transfection, cells were either mock infected or infected with PIV5 at a high m.o.i. (10 p.f.u. per cell). At 24 h p.i., the cells were fixed and immunostained for the viral NP protein (red) and the HA-tagged ISG56/IFIT1 (green).

Fig. 2.

ISG56/IFIT1 is primarily responsible for the IFN-induced changes in CPI− protein synthesis. Hep2/BVDV-N^pro (control) cells or Hep2/BVDV-N^pro.shISG56/IFIT1 (shISG56) cells were treated or not with IFN at 8 h prior to infection with a high m.o.i. of CPI− (IFN pre-treated), or were treated with IFN at 8 h p.i. (IFN post-treatment). At 20 h p.i., the cells were radioactively labelled with L-[³⁵S]methionine for 1 h and the viral proteins were immunoprecipitated. Total cell extracts (left-hand panels) and immunoprecipitates (right-hand panels) were separated by electrophoresis through a 4−12 % polyacrylamide gel and the labelled proteins visualized using a phosphoimager. The position of the M protein in the total cell extracts is indicated by asterisks. Samples of the total cell extracts corresponding to the samples in lanes 5–8 were also immunoblotted for the presence of STAT1 and ISG56/IFIT1 (bottom panel), which are inducible by IFN, and for β-actin.

Fig. 3.

Time course of expression of CPI− mRNA. Hep2/BVDV-N^pro cells were infected at a high m.o.i. (10 p.f.u. per cell) of CPI− and at various times p.i., the total cell RNA was extracted and subjected to qPCR for NP, P/V, M, HN and L mRNA. mRNA values are expressed as the quantity of the gene of interest relative to the quantity of β-actin mRNA. Results are shown as means±sem of duplicate samples in two separate experiments.

Fig. 4.

Analysis of the relative levels of CPI− mRNAs in Hep2/BVDV-N^pro and Hep2/BVDV-N^pro/shISG56/IFIT1-infected cells that were pre-treated (IFN pre) or not (−IFN) with IFN at 8 h prior to infection with CPI−, or were treated with IFN at 8 h p.i. (IFN post). At 20 h p.i., total cell RNA was extracted and the levels of NP, P/V, M, HN and L mRNA relative to β-actin mRNA were determined by qPCR. Results are shown as means±sem of duplicate samples in two separate experiments.

Fig. 5.

Comparisons of the relative amounts of viral proteins synthesized in infected cells and by in vitro translation of mRNA isolated from infected cells. Hep2/BVDV-N^pro cells were mock infected or infected with CPI− at a high m.o.i. and treated or not with IFN at 8 h p.i. (a) At 20 h p.i., the cells were radioactively labelled with [³⁵S]methionine for 1 h and the viral proteins immunoprecipitated. (b) At 20 h p.i., mRNA was isolated from parallel cultures and subject to in vitro translation using a rabbit reticulocyte lysate. Viral proteins were immunoprecipitated or not prior to being separated by electrophoresis through a 7–12 % gradient polyacrylamide gel, and labelled proteins were visualized using a phosphoimager. The position of the M protein in the total cell samples is indicated by asterisks.

Fig. 6.

In vitro ribose 2′-O-methylation assay. Incorporation of ³H into poly(A)-containing RNA from mock-infected cells or cells infected with PIV5 after in vitro 2′-O-methylation with recombinant vaccinia virus 2′-O-methyltransferase. In vitro-transcribed N7-methylated capped RNA (IVT-N-CM) was used as a positive control for the assay.

Fig. 7.

Viral cytoplasmic bodies do not form in response to IFN in cells in which ISG56/IFIT1 has been knocked down. Mock-infected or CPI−-infected A549/BVDV-N^pro (A549-N^pro) and A549/BVDV-N^pro.shISG56/IFIT1 (A549/N^pro.shISG56) cells were treated or not with IFN at 8 h p.i. Cells were fixed at 20 h p.i. and the distribution of the viral NP visualized by immunofluorescence.

Fig. 8.

(a) Yield of infectious CPI− virus following a low m.o.i. infection of naïve A549 (control) and A549/shISG56/IFIT1 (control/shISG56) cells. Monolayers of cells in 25 cm² flasks that had been pre-treated or not with IFN at 8 h prior to infection were infected with CPI− at 0.01 p.f.u. per cell and the amount of infectious virus in the culture medium at 48 h p.i. was determined by a plaque assay on Vero cells. (b) Relative plaque size of CPI− on naïve A549 cells (control), naïve A549/shISG56/IFIT1 (nA549/shISG56) and naïve A549/BVDV-N^pro cells (nA549). Monolayers of cells in six-well plates were infected with CPI−. At 7 days p.i., the cells were fixed and the virus plaques visualized by immunostaining the monolayers with a pool of mAbs to the NP and P proteins.