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Obligatory Structural Elements in Tobacco Etch Virus (TEV) RNA Required for Depurination by Pokeweed Antiviral Protein (PAP)
Author(s) -
Domashevskiy Artem,
Cheng Shu-Yuan
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.773.1
Subject(s) - depurination , rna , biology , antiviral protein , microbiology and biotechnology , ribosome , translation (biology) , messenger rna , ribosome inactivating protein , virology , viral replication , chemistry , biochemistry , virus , dna , gene
P okeweed a ntiviral p rotein (PAP) from P. americana is a r ibosome i nactivating p rotein (RIP) and is an N ‐glycosidase that removes specific purine residues from the s arcin/ r icin (S/R) loop of the large rRNA, arresting protein synthesis at the translocation step. PAP is a potent antiviral agent against many plant, animal, and human viruses. PAP inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNA and depurinates them at multiple sites downstream of the cap structure (Hudak, K. A., et al. RNA (2002) 8: 1148–59). This mechanism does not clarify inhibitory effect of PAP on the replication of uncapped viruses, and thus the overall mechanism of PAP antiviral activity remains to be elucidated. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe binding and enzymatic activities of PAP and its variants towards t obacco e tch v irus (TEV) RNAs, and thus understand the molecular basis behind PAP antiviral activity. The TEV RNA 5′‐leader is sufficient to confer cap‐independent translation, and contains an internal ribosome entry site (IRES) (Zeenko, V. and Gallie, D. R. J Biol Chem (2005) 280: 26813–24). We show that PAP possesses different binding and enzymatic domains within its structure, as well as that full 143‐nt TEV RNA leader sequence is required for PAP depurination of both m 7 GpppG‐capped and uncapped viral RNAs.