Katalin A. Hudak
PhD (Univ. of Waterloo)
Research Interests: Molecular Biology, Biochemistry, Plant Biology, Biotechnology
The Pokeweed plant (Phytolacca americana), a native of southern Canada and the USA, synthesizes a ribosome inactivating protein (RIP) that inhibits viral infection. Pokeweed antiviral protein (PAP) catalytically removes purine residues from a highly conserved stem-loop structure of the large rRNA, thereby blocking translation. PAP also displays broad-spectrum antiviral activity against several plant and animal viruses including Tobacco mosaic virus, Poliovirus and HIV. The mature form of this protein is extra-cellular and presumably enters the cell during viral infection, though the mechanism is unknown. Resistance has traditionally been attributed to an inhibition of protein synthesis, resulting in a lack of virus proliferation. However, recent findings show that antiviral activity can be maintained in the absence of host cell death, suggesting that PAP may target viral RNAs in addition to its well-understood substrate, the rRNA.
One of my areas of interest is to determine how PAP detects viral RNAs. In cell free systems, PAP binds to the 5’ cap structure present on some viral RNAs and depurinates these RNAs. However, not all viral RNAs known to be inhibited by PAP are capped. For example, Poliovirus contains an internal ribosome entry site (IRES), indicating that PAP may recognize other features of viral RNAs. By identifying the sequences of PAP that bind particular viral RNA structures, it may be possible to design a protein that specifically depurinates these RNAs.
Another aim of my research is to identify host proteins that interact with PAP. We know that PAP accesses rRNA by binding to L3, a highly conserved ribosomal protein involved in translation. In addition, cap recognition implies that PAP may play a broader role in the translation of messages during virus infection, one that likely involves the recruitment of other cellular factors. Combined genetic and biochemical approaches using yeast as a convenient system will identify host proteins that mediate the activity of PAP.
The results of both these projects will provide insight into the molecular mechanism by which PAP inhibits virus replication and will help us understand the function of this protein in regulating translation during viral infection. A practical application of these studies is the design of new antiviral agents, as few compounds presently exist that specifically target viruses.
Wang M and Hudak KA (2003) Applications of plant antiviral proteins. In: Genetic Engineering, Principles and Methods (J.K. Setlow, ed) vol. 25. Kluwer Academic Publishers, USA, in press.
Hudak KA, Bauman JD and Tumer NE (2002) Pokeweed antiviral protein binds to the cap structure of eukaryotic mRNA and depurinates the mRNA downstream of the cap. RNA 8: 1148-1159.
Hudak KA, Hammell AB, Yasenchak J, Tumer NE and Dinman JD (2001) A C-terminal deletion mutant of pokeweed antiviral protein inhibits programmed +1 ribosomal frameshifting and Ty1 retrotransposition without depurinating the sarcin/ricin loop of rRNA. Virology 279: 292-301.
Tumer NE, Hudak K, Di R, Coetzer C, Wang P, Zoubenko O (1999) Pokeweed antiviral protein and its applications. In: Current topics in microbiology and immunology (J Hammond, P McGarvey, V Yusibov, eds) vol. 240, Plant Biotechnology. Springer-Verlag, Berlin, pp 139-158.