Starting my second week of my internship today and after purifying my TCV sample last week, today I start on dissociation experiments.
I thought I would give a little background to my project today, as I haven't really explained what I am working on yet.
So my research project is called "Investigating the roles of sequence-specificity and packaging signals in the assembly of ssRNA plant virus capsids". The virus I am working on is called Turnip Crinkle Virus (TCV) and is a small, single-stranded, positive sense RNA virus.
I am working in the Stockley Lab at the University of Leeds, where I am currently a student studying Biochemistry.
The Stockley laboratory have recently developed single molecule fluorescence correlation spectroscopy (smFCS) as an assay for ssRNA virus reassembly in vitro at the nanomolar correlation concentrations of genome and coat protein (CP) found in vivo. Under these conditions they observed sequence-specificity in genome encapsidation, reflecting the situation in vivo. This specificity is driven by cognate interactions between multiple coat protein subunits and specific sequence/structural motifs in the genomic RNA that were termed packaging signals (PSs). These results overturn the currently widely-held paradigm that genomic RNAs are merely passengers in an electrostatically-driven assembly process controlled solely by CPs. These findings were presented in a recent paper written by A.Borodavka et al named “A two-stage mechanism of
viral RNA compaction revealed by single molecule fluorescence” (the paper can be found here http://www.ncbi.nlm.nih.gov/pubmed/23422316). They have used SELEX against viral CPs to identify putative PSs, including three major model ssRNA plant viruses; Turnip Crinkle Virus (TCV), Cowpea Chlorotic Mosaic Virus (CCMV) and Brome Mosaic Virus (BMV). The final aptamer pools were subjected to NextGen sequencing, revealing in each case conserved aptamer motifs within a number of sequence families. These sequences are putative PSs because when the genomic sequences were aligned there were statistically significant matches to the aptamers at multiple sites throughout the genomes. The vast majority of these PSs from all three viruses have predicted structures (via MFold), which encompass a stem-loop and exhibit suggestive conserved sequence motifs. They also contain the minimal assembly sequence, where interaction with CP is thought to repress translation of the helicase/replicase proteins. Modelling suggests that novel antiviral strategies targeting PS function should inhibit assembly.
These novel antiviral strategies are of great importance as plant viruses are a major problem in both the developed and third world. With billions of dollars’ worth of crops being destroyed by viruses every year this is a major threat to supporting the
growing world population.
My project aims to explore the molecular mechanism of capsid
formation and genome packaging of three simple plant virus models with the goal of potentially
uncovering new anti-viral strategies.
So far I have purified a sample of TCV from the plant leaves in which they are grown and concentrated it. The next thing I am trying to do, is the dissociate and re-assemble the RNA from its coat protein. These experiments replicate the experiments in the paper by P.K.Sorger, P.G.Stockley and S.C.Harrison named Structure and Assembly of Turnip Crinkle Virus, Mechanism of Reassembly in Vitro. I should, if everything goes to plan get a very nice graph, similar to the ones in the paper and get some nice electron microscope images of the virus I am working on, which I will post on here once I have them.
Well today has been a pretty sciency post! You may have enjoyed it, or not, but most of my other posts will not be as full on!