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Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR.

ABSTRACT: Ranaviruses (family Iridoviridae) are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted protein, designated vIF2?, which shows homology with the eukaryotic translation initiation factor 2?. In analogy to distantly related proteins found in poxviruses vIF2? might act as an inhibitor of the antiviral protein kinase PKR.We have characterized the function of vIF2? from Rana catesbeiana virus Z (RCV-Z). Multiple sequence alignments and secondary structure prediction revealed homology of vIF2? with eIF2? throughout the S1-, helical- and C-terminal domains. Genetic and biochemical analyses showed that vIF2? blocked the toxic effects of human and zebrafish PKR in a heterologous yeast system. Rather than complementing eIF2? function, vIF2? acted in a manner comparable to the vaccinia virus (VACV) K3L protein (K3), a pseudosubstrate inhibitor of PKR. Both vIF2? and K3 inhibited human PKR-mediated eIF2? phosphorylation, but not PKR autophosphorylation on Thr446. In contrast the E3L protein (E3), another poxvirus inhibitor of PKR, inhibited both Thr446 and eIF2? Ser51 phosphorylation. Interestingly, phosphorylation of eIF2? by zebrafish PKR was inhibited by vIF2? and E3, but not by K3. Effective inhibition of PKR activity coincided with increased PKR expression levels, indicative of relieved autoinhibition of PKR expression. Experiments with vIF2? deletion constructs, showed that both the N-terminal and helical domains were sufficient for inhibition of PKR, whereas the C-terminal domain was dispensable.Our results show that RCV-Z vIF2? is a functional inhibitor of human and zebrafish PKR, and probably functions in similar fashion as VACV K3. This constitutes an important step in understanding the interaction of ranaviruses and the host innate immune system.

SUBMITTER: Rothenburg S

PROVIDER: S-EPMC3068933 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR.

Rothenburg Stefan S Chinchar V Gregory VG Dever Thomas E TE

BMC microbiology 20110318

<h4>Background</h4>Ranaviruses (family Iridoviridae) are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted protein, designated vIF2α, which shows homology with the eukaryotic translation initiation factor 2α. In analogy to distantly related proteins found in poxviruses vIF2α might act as an inhibitor of the antiviral protein kinase PKR.<h4>Results</h4>We have characterized the func ...[more]

PMID: 21418572

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Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I interferon (IFN-?/?) response, the protein kinase R (PKR)-mediated stress response is being recognized as an important innate response pathway. Upon detecting viral dsRNA, PKR phosphorylates eIF2?, leading to the inhibition of cellular and viral translation and the formation of stress granules (SGs), which are increasingly recognized as platforms for antiviral signaling pathways. It is unknown whether cellular infection by MERS-CoV activates the stress response pathway or whether the virus has evolved strategies to suppress this infection-limiting pathway. Here, we show that cellular infection with MERS-CoV does not lead to the formation of SGs. By transiently expressing the MERS-CoV accessory proteins individually, we identified a role of protein 4a (p4a) in preventing activation of the stress response pathway. Expression of MERS-CoV p4a impeded dsRNA-mediated PKR activation, thereby rescuing translation inhibition and preventing SG formation. In contrast, p4a failed to suppress stress response pathway activation that is independent of PKR and dsRNA. MERS-CoV p4a is a dsRNA binding protein. Mutation of the dsRNA binding motif in p4a disrupted its PKR antagonistic activity. By inserting p4a in a picornavirus lacking its natural PKR antagonist, we showed that p4a exerts PKR antagonistic activity also under infection conditions. However, a recombinant MERS-CoV deficient in p4a expression still suppressed SG formation, indicating the expression of at least one other stress response antagonist. This virus also suppressed the dsRNA-independent stress response pathway. Thus, MERS-CoV interferes with antiviral stress responses using at least two different mechanisms, with p4a suppressing the PKR-dependent stress response pathway, probably by sequestering dsRNA. MERS-CoV p4a represents the first coronavirus stress response antagonist described.

Project description:Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2? and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as ?-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and ?-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and ?-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells.Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and ?-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.

Dataset Information

Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR.

Publications

Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR.

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