Abstract
Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.
Highlights
Viral infection triggers activation of the integrated stress response (ISR)
The NSs with a C-terminal FLAG tag (NSs::FLAG) should retain its PKR-evading properties while tagging at the N-terminus (FLAG::NSs) blocks this functionality[29]. These constructs were genomically integrated into our previously generated ISR reporter system, in which both changes in ATF4 translation and general translation can be monitored[5]. Both NSs::FLAG and FLAG::NSs were stably expressed in these cells without impacting the levels of key ISR components (Fig. 1a)
As one of the strategies in the evolutionary arms race between viruses and the host cells they infect, mammalian cells activate the ISR to temporarily shut down translation, preventing the synthesis of viral proteins
Summary
Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. 1234567890():,; The integrated stress response (ISR) is a conserved eukaryotic stress response network that, upon activation by a diverse set of stressors, profoundly reprograms translation It is coordinated by at least four stress-responsive kinases: PERK (responsive to protein misfolding in the endoplasmic reticulum), PKR (responsive to viral infection), HRI (responsive to heme deficiency and oxidative and mitochondrial stresses), and GCN2 (responsive to nutrient deprivation)[1,2,3,4]. Under non-stress conditions, eIF2 engages eIF2B through multiple interfaces along a path spanning the heterodecamer In this arrangement, eIF2α binding to eIF2B critically positions the GTPase domain in eIF2’s γ-subunit, allowing for efficient catalysis of nucleotide exchange. I-State eIF2B(αβδγε)[2] exhibits enzymatic activity and substrate engagement akin to the tetrameric eIF2Bβδγε subcomplex; eIF2-P inhibition of eIF2B converts the decamer into conjoined tetramers, which reduces its GEF activity, lowers the cell’s TC concentration, and results in ISR-dependent translational reprogramming[5,6]
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