Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

Abstract

The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2␣ phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2␣ by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2␣ phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2␣ phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5 untranslated region (5UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5UTR and IGR IRES.

Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

“…1D). The stimulation of translation by CrPV-1A is unexpected, since CrPV infection in S2 cells results in a nearly complete inhibition of host translation (49,60,61). To address whether expression of CrPV-1A affected IRES translation, we transfected an in vitrotranscribed bicistronic CrPV minigenome RNA (Fig.…”

“…CrPV infection modulates a number of cellular pathways, including host translation, transcriptional shutoff, and SG assembly (48,49,60,62,64). CrPV encodes at least 12 viral proteins, including 8 nonstructural and 4 structural proteins (61) (Fig.…”

“…Intracellular virus, which was collected from multiple freeze-thawing cycles of the transfected cells, was then used to reinfect naive cells to expand the virus stock, as described previously (60). Viral titer was determined by focus-forming assay (60). For mutant R146A virus, virus preparations were RT-PCR amplified and sequenced to verify that the mutation did not revert.…”

“…Cells were transfected with CrPV-3 or CrPV-3(R146A) RNA clone as described in reference 65. Intracellular virus, which was collected from multiple freeze-thawing cycles of the transfected cells, was then used to reinfect naive cells to expand the virus stock, as described previously (60). Viral titer was determined by focus-forming assay (60).…”

“…Transfer of RNA was confirmed by methylene blue staining. Northern blotting was performed as described previously (60).…”