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J. Biol. Chem., Vol. 280, Issue 20, 20069-20075, May 20, 2005

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Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells^*

Daniel Picard, C. Cheng Kao ¶, and Katalin A. Hudak||

From the Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada and the Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843-2128

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.

Received for publication, November 30, 2004 , and in revised form, March 7, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ C. Kao acknowledges funding from the National Science Foundation.

^|| K. Hudak is grateful for funding support from the Natural Sciences and Engineering Research Council of Canada. To whom correspondence should be addressed: Dept. of Biology, York University, 4700 Keele St., Toronto, Ontario M3J 1P3, Canada. Tel.: 416-736-2100 (ext. 33470); Fax: 416-736-5698; E-mail: hudak{at}yorku.ca.

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