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Plants produce hundreds of defense compounds essential for their survival. These factors help them respond to environmental cues and fend off pathogens. My lab is focused on understanding the function of a specific group of enzymes called RNA glycosidases. These enzymes hydrolyse purine bases from certain RNAs, including ribosomal RNA and viral RNAs. The goal of our research is to understand the regulation and activity of a glycosidase synthesized by the pokeweed plant, Phytolacca americana, called pokeweed antiviral protein (PAP).

The antiviral activity of PAP was first documented in 1927 and subsequent biochemical studies showed that it removed an adenine base from the sarcin/ricin loop of the 28S rRNA, exactly like the notorious toxin, ricin.  Both proteins inhibited translation in vitro because damage to the rRNA prevented elongation factor binding to the ribosomes. One possibility to explain how PAP could function as a defense protein was to kill cells infected by virus. However, we and others have since shown that PAP maintains its antiviral activity without signs of toxicity.  The absence of host cell death suggested that PAP could directly affect viruses by depurinating their genomic RNA and that PAP is likely a component of a larger network of responses by the plant to pathogens.

Our two current research areas are:

1) Characterization of pokeweed defense pathways and regulation of PAP

We have shown recently that expression of PAP increases with jasmonic acid, at the mRNA and protein levels. Moreover, we have identified a small RNA, also responsive to jasmonic acid, which targets the antiviral mRNA for cleavage (Klenov et al., 2015). Jasmonic acid is a plant hormone important in defense against necrotrophic pathogens and insect herbivores. In light of these results, we have sequenced the transcriptome and small RNA pool of pokeweed, with and without jasmonic acid stimulation.

Our goal is to determine the effects of jasmonic acid on the pokeweed transcriptome, to investigate the role of this hormone in regulating the antiviral protein, and to identify defense genes and pathways important in this response. These analyses will provide us with new information on how pokeweed defends itself against pathogens. From a broader perspective, a wealth of genomic knowledge remains unknown in this species, as large-scale sequencing projects have not been reported for pokeweed or any other members of its plant family.

This knowledge will help us to improve pathogen resistance in crop plants (a close relative of pokeweed is the sugar beet). Concern over climate change and its impact on food production have increased our need to develop crops plants better able to survive stress. This bioinformatics-centered approach represents the foundation of a new direction of research in our lab for study of defense-related gene expression. Though basic in nature, our research will contribute to strengthening agriculture, which will continue to be influenced by environmental change.

 

2) Mechanism of action against viruses

We have made substantial advances in understanding how PAP inhibits various stages of a virus lifecycle. A summary of our specific findings is below:

PAP’s effect on Brome mosaic virus (BMV)

  • PAP expression in barley cells inhibits the replication of BMV, a plant virus that infects barley.  PAP depurinates the viral RNA, which prevents the viral replicase from synthesizing new copies of viral RNA because the replicase stalls at the abasic sites (Picard et al., 2005).
  • Depurination of BMV RNA also inhibits viral protein synthesis because ribosomes stall at abasic sites during elongation.  We also showed that for BMV RNA, this stalling triggered degradation of the RNA by the No-go decay pathway (Gandhi et al., 2008).
  • The third effect of BMV RNA depurination was observed on the formation of viral particles.  Particles are comprised of viral coat protein bound to viral RNA and we showed that coat protein does not bind to depurinated RNA, which significantly reduces particle production (Karran and Hudak, 2011).

PAP’s effect on Human T-cell Leukemia Virus (HTLV-1) and Human immunodeficiency virus (HIV-1)

  • PAP expression inhibits the replication of HTLV-1 from a proviral clone.  PAP substantially decreased viral protein synthesis, including the synthesis of Tax, a viral transactivator protein, which resulted in decreased transcription of genomic RNA from the viral promoter (Mansouri et al., 2009).
  • PAP also inhibits the replication of HIV-1 and significantly reduces viral protein synthesis.  By depurinating Vif RNA, PAP diminishes the amount of Vif protein synthesized. As a result, the host restriction factor APOBEC3G is not targeted by Vif and can hypermutate the viral genome and reduce infectivity. Therefore, PAP indirectly enhances the innate immunity of cells against HIV-1 (Krivdova and Hudak, 2015).
  • The activity of viral protein Rev, which is involved in processing of viral RNAs, is also inhibited in cells expressing PAP.  Reduced Rev activity results in altered splicing ratio among the viral mRNAs, which prevents production of particles because the relative amounts of viral structural to regulatory proteins is disrupted (Zhabokritsky et al., 2014).