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Texas A&M's Herman Scholthof on Viral Inhibition of PTGS



Name: Herman Scholthof

Position: professor, Texas A&M University

Background: Postdoc, University of California, Berkeley — 1990-1994

PhD, molecular virology, University of Kentucky — 1990

MS, plant virology, Wageningen University — 1986

BS, plant pathology, Wageningen University — 1984

At Texas A&M University, Herman Scholthof researches the molecular mechanisms that affect a plant's resistance or susceptibility to viruses. One of these mechanisms involves the protein P19, which appears to play a role in a virus' ability to inhibit post-transcriptional gene silencing.

Recently, Scholthof spoke with RNAi News about his work and a recently awarded National Institute of Allergy and Infectious Diseases-funded project to characterize RISC.

Let's begin with an overview of your lab.

The model system we are mostly using is called tomato bushy stunt virus. It's a virus that we like a lot because it's very easy to work with: It infects a lot of plants … but there is no biological vector — an insect, for example — known to transmit this virus so it doesn't spread that fast. For our work, this is a good safety feature.

This virus is a very good model system for what I refer to as positive-sense RNA viruses, which are viruses that have an RNA genome that is essentially immediately ready for translation when it enters a cell. TBSV really accumulates rapidly and to large amounts … and [so] it's very easy to detect and work with.

Compared to animal viruses or human viruses, plant viruses have to overcome an additional obstacle. Plants don't have a skeleton; [instead] each cell has a thick cell wall … and the openings between these cells are generally too small for a virus to go through. Our main interest is how plant viruses do this.

In my lab, we look at what we call cell-to-cell movement. How does a virus, once it enters a single cell, which it can do … [by entering through damaged areas] … There are two ways: It can either make itself smaller or make the opening [between cells] bigger … [and] they do both.

We know the virus cannot do all this by itself; most viral functions need host elements and host proteins to complete whatever biochemical activity they need to perform. Our main interest is what these host factors are. We have some candidates that we think the virus piggybacks on and that know the way to the plasmodesmata.

That's one part of our research. But we also had a question about … long-distance spread that is necessary to achieve systemic infections. We knew from previous work that there … are proteins important in this process, and for awhile we have been working on one of these proteins, which we refer to as P19. We have the virus genome available … in a DNA plasmid, and we can manipulate it any way we want and make RNA back from it to see if it's still infectious. By using this infectious clone, we were able to demonstrate that P19 was necessary for … systemic spread within several plant species.

We were dealing with the question, 'Is P19 an active component in promoting spread or is it perhaps inactivating or interfering with certain defenses plants may come up with?' We don't know the exact answer yet — we're looking at different angles and this protein always throws different surprises at us, and I think it probably has multiple activities depending on the plant that it's in.

Then we were able to confirm that this P19 protein is what is referred to as a suppressor of RNA gene silencing. We were interested in finding out how this protein interferes with this silencing. We were helped by studies that others performed where they resolved the structure of the P19 protein, and it appeared that P19 had the ability to specifically bind siRNAs — and we then showed that in plants it sort of sweeps them up so they would not be available for RISC. Therefore the viral genome would not be attacked by [the plant's] silencing system and a systemic infection could proceed.

Our model was that this P19 protein, at least in some plants, rather than being directly involved in promoting virus movement in the plant, it actually was counteracting a defense system by preventing silencing from becoming too widely spread through the plant.

We then asked the question, 'Can we find this RISC activity in the presence of P19 protein?' Our hypothesis would be, 'No, because P19 would prevent RISC from becoming activated.' The other side of the hypothesis was, 'If this virus gets in and does not make P19, RISC would become activated and would destroy the viral genome.'

This has not been demonstrated for any virus system, and it's clear we need to understand more about how this silencing actually works because siRNAs are being explored as therapeutic agents, [and] a problem that's being encountered is non-specific effects. Clearly, a better biochemical understanding of how silencing operates would be a benefit.

We set out to do this and are still working on it. It's a very tedious process because these macromolecules we work with are very fragile and they lose activity very fast. But in a nutshell what we have found … is that in absence of P19 we were able to isolate molecular complexes from plants that have RISC activity, whereas in presence of P19 this activity was not detected. To demonstrate this activity we developed a system whereby we fed viral RNA in a test tube to plant extracts thought to contain these [complexes] to see if the viral RNAs would be targeted for cleavage, and on the whole this works very effectively. This system was developed by Rustem Omarov, an associate research scientist in the lab.

The NIH grant we have [is supporting research focused on] finding out the protein composition of this complex that shows this RISC activity, [as well as determining if] there is something unique to our system. In other words, in order for this to be somewhat useful, we need to be sure we're going after some general phenomenon. So we're now testing if other plant viruses activate a similar RISC defense system in plants. Can we show the same thing in vitro when we isolate these high-molecular weight complexes from plants, and do we see the same RNA cleavage activity?

One thing I forgot to mention is that this cleavage activity that we do in vitro is sequence-specific, and that's a hallmark for RISC. And there are some other biochemical features associated with it — co-factors that it needs, et cetera — that all are in line with what people know so far about RISC.

We think we have the real thing, and the reviewers of the grant agreed. So that's now our main goal: to biochemically characterize this RISC that we have at the protein level, and then see if other plant viruses other than the model system we work with also induce a similar RISC activity in plants.

What do you see as the long-term impact of your research?

I hope that our work leads to a better fundamental understanding of how RISC operates and that others can implement this knowledge in the future for improved design and administering of therapeutic siRNAs, or otherwise activate antiviral RISC, and to minimize off-target effects