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EIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

ABSTRACT: RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2?. Phosphorylation of the ? subunit converts the eIF2??? complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2?. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2? phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2? binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2?.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2?, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2? or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2?. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

SUBMITTER: Wuerth JD

PROVIDER: S-EPMC7360930 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

Wuerth Jennifer Deborah JD Habjan Matthias M Kainulainen Markus M Berisha Besim B Bertheloot Damien D Superti-Furga Giulio G Pichlmair Andreas A Weber Friedemann F

mBio 20200714 4

RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome bin ...[more]

PMID: 32665273

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Project description:African swine fever virus (ASFV) is one of the most contagious and lethal viruses infecting pigs. This virus is endemic in many countries and has very recently spread to China, but no licensed vaccines or treatments are currently available. Despite extensive research, the basic question of how ASFV-encoded proteins inhibit host translation remains. Here, we examined how ASFV interfered with host translation and optimized viral gene expression. We found that 14 ASFV proteins inhibited Renilla luciferase (Rluc) activity greater than 5-fold, and the protein with the strongest inhibitory effect was pE66L, which was not previously reported. Combined with bioinformatical analysis and biochemical experiment, we determined that the transmembrane (TM) domain (amino acids 13-34) of pE66L was required for the inhibition of host gene expression. Notably, we constructed a recombinant plasmid with the TM domain linked to enhanced green fluorescent protein (EGFP) and further demonstrated that this domain broadly inhibited protein synthesis. Confocal and biochemical analyses indicated that the TM domain might help proteins locate to the endoplasmic reticulum (ER) to suppress translation though the PKR/eIF2α pathway. Deletion of the E66L gene had little effect on virus replication in macrophages, but significantly recovered host gene expression. Taken together, our findings complement studies on the host translation of ASFV proteins and suggest that ASFV pE66L induces host translation shutoff, which is dependent on activation of the PKR/eIF2α pathway.Importance African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA virus superfamily that predominantly replicates in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from approximately 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames (ORFs), of which half the encoded proteins have not been explored. Our study showed that 14 proteins had an obvious inhibitory effect on Renilla luciferase (Rluc) gene synthesis, with pE66L showing the most significant effect. Furthermore, the transmembrane (TM) domain of pE66L broadly inhibited host protein synthesis in a PKR/eIF2a pathway-dependent manner. Loss of pE66L during ASFV infection had little effect on virus replication, but significantly recovered host protein synthetic. Based on the above results, our findings expand our view of ASFV in determining the fate of host-pathogen interactions.

Dataset Information

EIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

Publications

eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

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