The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling

作者：Emmely E. Treffers, Ali Taş, Florine E. M. Scholte, Arnoud H. de Ru, Eric J. Snijder, Peter A. van Veelen, Martijn J. van Hemert · 发表于：PLoS Pathogens · 年份：2023 · DOI：10.1371/journal.ppat.1011179 · 被引用次数：19 · 研究领域：Mosquito-borne diseases and control、HIV Research and Treatment、Viral Infections and Outbreaks Research

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, ...