Translational control by influenza virus. Selective and cap-dependent translation of viral mRNAs in infected cells.

M.S Garfinkel,M.G Katze

doi:10.1016/s0021-9258(19)50435-5

Abstract

In cells infected by influenza virus type A, host protein synthesis undergoes a rapid and dramatic shutoff. To define the molecular mechanisms underlying this selective translation, a transfection/infection protocol was developed utilizing viral and cellular cDNA clones. When COS-1 cells were transfected with cDNAs encoding nonviral genes and subsequently infected with influenza virus, protein expression from the exogenous genes was diminished, similar to the endogenous cellular genes. However, when cells were transfected with a truncated influenza viral nucleocapsid protein (NP-S) gene, the NP-S protein was made as efficiently in influenza virus infected cells as in uninfected cells, showing that the NP-S mRNA, although expressed independently of the influenza virus replication machinery, was still recognized as a viral and not a cellular mRNA. Northern blot analysis demonstrated that the selective blocks to nonviral protein synthesis were at the level of translation. Moreover, polysome experiments revealed that the translational blocks occurred at both the initiation and elongation stages of cellular protein synthesis. Finally, we utilized this transfection/infection system as well as double infection experiments to demonstrate that the translation of influenza viral mRNAs probably occurred in a cap-dependent manner as poliovirus infection inhibited influenza viral mRNA translation.

Highlights

In cells infected by influenzavirus type A, host pro- Interestingly it was recently shown that dephosphorylation of tein synthesis undergoes a rapid and dramatic shutoff.another component of the cap binding protein complex, eIF
The inhibition of cellular protein synthesis inpoliovirus infected cells is correlated with the degradation of the 220,000-dalton protein, P220, a component of the cap binding protein complex, eulated in adenovirus-infected cells (Katze et al, 1984, 1986a, 198613).In other experiments we have shown that aninfluenza viral nucleocapsid protein (NP) protein expressed by a recombinant adenovirus escaped the adenovirus imposed blocks on translation and was synthesized as efficiently as incells doubly infected with adenovirus and influenza virus (Alonso-Caplen et al.,1988)
To confirm that the structureof viral mRNAs is importantfor selective translation, we designed an in uiuo assay in which we could follow thetranslational fate of exogenous cellular and influenza genes during influenza virus infection (Fig. hi).By transfection analysis, we introduced cellular genes the products of which could be distinguished from endogenous cellular gene products

Summary

RESULTS

Introduced CellularGene Expression Is Blocked at the Initiation and Elongation Steps of Protein Synthesis in Influenza Virus-infected Cells-Previouswork has demonstrated that influenza virus invoked a translational control mechanism as part of its overall strategy to maximize influenza viral gene expression during infection. A combined initiation-elongation block (which was previously determined to occur on endogenous cellular mRNA translation in influenza virus infected synthesized SEAPa,s measured by enzymatic activity, cells; Katze et al, 1986a)would result in asignificant propordropped 5-10-fold over the course of influenza viral infection tion of the mRNAs remaining associated with polysomes (Fig. 3A).We found identical reductions in thelevels of newly which probably would not increase in size, and some mRNAs synthesized SEAP protein by pulse-labeling with [35S]methi- would be on smaller polysomes or displaced from polysomes. To mincipitating the radiolabeled extracts with an IL-2-specific an- imize artifactual sticking of RNA to polysomes, 500mMKC1 tibody which recognizes both the glycosylated and unglyco- was included in the sucrose gradients; 500 mM KC1also should sylated forms of the protein (Fig. 3 B ) Quantitation of this have caused most 80 S monosomes to dissociate into 60 and decline by laser densitometryscanning revealed that IL-2 40 S ribosomal subunits (Blobeland Sabatini,1971).As shown. AI 39 hours post-transfection, infectwith 50 pfukell influenza virus or poliovirus

B HPI 5 r!

B P3 P3 P2 M3 HPI

DISCUSSION