No abstract

Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler’s virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.

The leader protein of cardioviruses, Theiler's murine encephalomyelitis virus (TMEV) and encephalomyocarditis virus (EMCV), is a multifunctional protein known to antagonize type I interferon expression and to interfere with nucleocytoplasmic trafficking of host proteins and mRNA. This protein plays an important role in the capacity of TMEV to establish persistent infection of the central nervous system. Mutant forms of the TMEV leader protein were generated by random mutagenesis and selected after retroviral transduction on the basis of the loss of the highly toxic nature of this protein. Theiler's murine encephalomyelitis virus (TMEV) is a neurotropic picornavirus that belongs to the Cardiovirus genus (reviewed by Brahic et al. [5]). The leader (L) protein of TMEV is a short (76 amino acids), very acidic protein. This protein helps the establishment of persistent TMEV infections in the central nervous system by antagonizing innate host defenses. It inhibits the transcription of type I interferon (IFN) and selected cytokine and chemokine genes, likely through inhibition of IRF-3 dimerization (19,25,28,33,34). It also interferes with nucleocytoplasmic trafficking of cellular proteins and blocks mRNA export from the nucleus (11,28). These activities correlate with the phosphorylation of nucleoporin 98 (Nup98) (28).The sequence of the L protein contains three domains: a zinc finger domain that was shown to bind divalent cations (7), an acidic central domain, and a Ser/Thr-rich domain (see Fig. 2). The L protein encoded by encephalomyocarditis virus (EMCV) shows 35% amino acid identity with the TMEV L protein. In the EMCV L protein, the zinc finger and the acidic domain are conserved but the C-terminal region encompassing the Ser/Thr-rich domain is lacking. In spite of this difference, L proteins of cardioviruses share the abilities to antagonize IFN production, to affect nucleocytoplasmic trafficking of mRNA and proteins, and to promote nucleoporin hyperphosphorylation (3,16,21,(25)(26)(27)36).Likely as a consequence of mRNA nuclear export inhibition, the TMEV L protein mediates shutoff of host protein synthesis and is very toxic when expressed in cells (2,11,28). In this work, we took advantage of this toxicity to select L mutants that lost the ability to shut off host protein synthesis in order to identify critical domains of the L protein and to test whether the multiple activities of the L protein can be uncoupled. MATERIALS AND METHODS Cells and viruses.BHK-21 cells were cultured as previously described (34). BALB/3T3, L929, and Phoenix-Eco cells were cultured in Dulbecco's modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (MP Biologicals), 100 IU of penicillin/ml, and 100 g of streptomycin/ml. Phoenix-Eco cells were kindly provided by G. Nolan via the ATCC (SD-3444).TMEV derivatives were produced by electroporation of BHK-21 cells (23) with genomic RNA transcribed in vitro from plasmids carrying the corresponding cDNAs. Virus DA1 was produced from plasmid pTMDA1 (10,22,23). Virus TM598 i...

Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler’s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA. IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler’s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.