Leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation

Cheryl M.T. Dvorak, David J. Hall, Marchel Hill, Michael Riddle, Andrew Pranter, Johnathan Dillman, Michael Deibel, Ann C. Palmenberg

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

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 elF-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.

Original languageEnglish (US)
Pages (from-to)261-271
Number of pages11
JournalVirology
Volume290
Issue number2
DOIs
StatePublished - Nov 25 2001
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health Grant AI-17331 to A.C.P. and predoctoral training Grant T32-GM07215 to C.M.T.D. The authors thank the Holcomb Research Institute and Butler University for providing support and facilities that aided in this work and Joe Binder for critical reading of the manuscript.

Keywords

  • Cardiovirus
  • Enzyme purification
  • Picornavirus
  • Translation

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