Eukaryotic Translation Initiation Factor 4F Architectural Alterations Accompany Translation Initiation Factor Redistribution in Poxvirus-Infected Cells

Abstract

Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.

PI3K Signaling Regulates Rapamycin-Insensitive Translation Initiation Complex Formation in Vaccinia Virus-Infected Cells

“…Although this increase in repressor abundance may be directly linked to the degree to which 4E-BP1 becomes hypophosphorylated, and thus stabilized in these samples, the precise mechanism by which PI3K inhibition exerts these effects remains to be determined. In addition, phosphorylation of the cap-binding protein eIF4E, known to enhance VV replication (28), was also suppressed in LY294002-treated cultures (Fig. 2B).…”

“…At 16 h p.i., cultures were photographed by phase microscopy. 28,30). Although this increase in repressor abundance may be directly linked to the degree to which 4E-BP1 becomes hypophosphorylated, and thus stabilized in these samples, the precise mechanism by which PI3K inhibition exerts these effects remains to be determined.…”

“…VV has been shown to promote the formation of eIF4F complexes and inactivate the translational repressor protein 4E-BP1 in a rapamycin-sensitive manner (28), but how this is accomplished and whether these two events are linked in VV-infected cells remain unknown. Activation of signaling upstream of mTOR in cells infected with myxoma virus, a rabbitspecific poxvirus, has been shown to involve direct stimulation of Akt by the host range factor MT-5, and this occurs in a PI3K-independent manner (26,32,33).…”

“…Like all viruses, in the absence of its own translational machinery vaccinia virus (VV), a prototypical poxvirus (17), has evolved a range of strategies to prevent the host antiviral response from shutting down translation and to commandeer the cellular protein synthesis machinery to serve its own ends (4,8,12,24,28). VV has been shown to promote the formation of eIF4F complexes and inactivate the translational repressor protein 4E-BP1 in a rapamycin-sensitive manner (28), but how this is accomplished and whether these two events are linked in VV-infected cells remain unknown.…”

“…NHDFs were either mock infected or infected in the presence of LY294002, rapamycin, or a dimethyl sulfoxide (DMSO) solvent control. At 16 h p.i., soluble cell extracts were prepared and cap-binding eIF4E was recovered by 7-methyl GTP batch chromatography (28). Samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with antisera against eIF4E, eIF4G, and 4E-BP1 to determine the composition of eIF4E-bound complexes in each sample ( Fig.…”