Leader Protein of Encephalomyocarditis Virus Binds Zinc, Is Phosphorylated during Viral Infection, and Affects the Efficiency of Genome Translation

Abstract

Encephalomyocarditis virus (EMCV) is the prototype member of the cardiovirus genus of picornaviruses. For cardioviruses and the related aphthoviruses, the first protein segment translated from the plus-strand RNA genome is the Leader protein. The aphthovirus Leader (173-201 amino acids) is an autocatalytic papain-like protease that cleaves translation factor eIF-4G to shut off cap-dependent host protein synthesis during infection. The less characterized cardioviral Leader is a shorter protein (67-76 amino acids) and does not contain recognizable proteolytic motifs. Instead, these Leaders have sequences consistent with N-terminal zinc-binding motifs, centrally located tyrosine kinase phosphorylation sites, and C-terminal, acid-rich domains. Deletion mutations, removing the zinc motif, the acid domain, or both domains, were engineered into EMCV cDNAs. In all cases, the mutations gave rise to viable viruses, but the plaque phenotypes in HeLa cells were significantly smaller than for wild-type virus. RNA transcripts containing the Leader deletions had reduced capacity to direct protein synthesis in cell-free extracts and the products with deletions in the acid-rich domains were less effective substrates at the L/P1 site, for viral proteinase 3Cpro. Recombinant EMCV Leader (rL) was expressed in bacteria and purified to homogeneity. This protein bound zinc stoichiometrically, whereas protein with a deletion in the zinc motif was inactive. Polyclonal mouse sera, raised against rL, immunoprecipitated Leader-containing precursors from infected HeLa cell extracts, but did not detect significant pools of the mature Leader. However, additional reactions with antiphosphotyrosine antibodies show that the mature Leader, but not its precursors, is phosphorylated during viral infection. The data suggest the natural Leader may play a role in regulation of viral genome translation, perhaps through a triggering phosphorylation event.

Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase

“…This inhibition happens in infected cells even before the virus begins to replicate (6,26). The cascade involves Erk1/2 and p38 kinases and is absolutely dependent on Ran:L E interactions and also on the dual phosphorylation of L E itself, an activity that is a prerequisite, and not a consequence, of the Nup modifications (7,10,15,16,27). Furthermore, the L E zinc finger motif must remain intact and chelated to the metal for the protein to function (11).…”

Nucleocytoplasmic Traffic Disorder Induced by Cardioviruses

“…Although the exact role and mechanism(s) of the leader protein action in infected cells remain unknown, the protein has been implicated in several important aspects of the viruscell interaction, such as control of viral (24) and host (81) translation as well as suppression of interferon production (19,FIG. 11.…”

“…To elucidate which structural elements of the L protein might be required to make the nuclear envelope permeable, several additional mengovirus mutants were created and tested. The L protein contains a predicted Zn finger near its N terminus (16,24) and is phosphorylated at Thr47 (82). A mutant with the Zn finger domain of L disturbed by two engineered substitutions (Cys193Ala and Cys223Ala) was unable to induce relocation of the nuclear marker ( Fig.…”

Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus

“…The cardiovirus leader polypeptide binds zinc, is phosphorylated during infection and plays a role in the regulation of viral genome translation (Dvorak et al, 2001). The functions of the kobuvirus, teschovirus and sapelovirus leaders are not known.…”