Molecular Analysis of Pokeweed Antiviral Protein Gene Function

Project Details

Description

9419919 Tumer Pokeweed antiviral protein (PAP) is a ribosome inactivating protein (RIP) of 29,000 daltons that is extracted from the leaves of Phytolacca americana (Pokeweed). RIPs are site-specific RNA N-glycosidases, which catalytically remove a single adenine from a conserved loop of the 28S rRNA of eukaryotic ribosomes. This lesion interferes with elongation factor-2 (EF-2) binding and blocks protein synthesis at the translocation step. PAP was discovered due to its ability to inhibit the transmission of tobacco mosaic virus (TMV) in plants and it was subsequently demonstrated that the purified protein is equally effective against a number of other plant viruses and animal viruses, including HIV. Recently, we cloned the cDNA encoding PAP and demonstrated that expression of PAP in transgenic plants leads to broad spectrum resistance to viral infection (Proc. Natl. Acad. Sci. USA 90: 7089-7093, 1993). The primary objective of this to generate critical information regarding the underlying mechanism for the antiviral activity of PAP. We will test the hypothesis that the antiviral activity of PAP is distinct from its ribosome inhibitory activity by isolating nontoxic PAP mutants using general mutagenesis and selection in Saccharomyces cerevisiae. PAP mutants will be characterized (i) molecularly by sequence analysis and (ii) biologically by measuring their enzymatic activity and antiviral activity. We will determine if mutations that render PAP nontoxic are located at the active site or at other domains and whether the residues that are involved in enzymatic activity are critical for antiviral activity. These studies will provide insight into the underlying mechanism of action of PAP. %%% Pokeweed antiviral protein (PAP) is a ribosome inactivating protein that is extracted from the leaves of Phytolacca americana (Pokeweed). PAP catalytically removes a single adenine from 28S rRNA thereby inactivating the ribosome and inhibiting protein synthesis. PAP was discovered due to its ability to i nhibit the transmission of tobacco mosaic virus in plants, and was subsequently demonstrated to be equally effective against a number of other plant and animal viruses, including HIV. The primary objective of this research is to determine the mechanism for the antiviral activity of PAP, and to test the hypothesis that the antiviral activity of PAP is distinct from its ribosome inhibitory activity. These studies will provide critical insight into the underlying mechanism of action of PAP. *** ??

StatusFinished
Effective start/end date3/15/952/28/98

Funding

  • National Science Foundation: $99,474.00

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