Structural and functional studies in lentivirus RNA encapsidation

Lead Research Organisation: University of Cambridge
Department Name: Medicine

Abstract

There is no vaccine for HIV/AIDS. Drug combinations slow the virus but it has developed resistance to all the current ones and new ones are needed which stop virus replication. When HIV assembles it packages its genes, which are on a long piece of RNA similar to our own DNA. There are lots of other RNAs in the cell and HIV recognises its own because this RNA folds up into a complex but distinct ?knot? like shape. The virus captures this RNA at a particular place inside the cell. Drugs blocking this process would provide a potentially very powerful new weapon against HIV. The plan is to deduce the structures of all the molecules involved and also where the capture happens so we can design antiviral molecules and get them to the correct place to work against the virus. The work will also have spin off benefits for gene therapy.

Technical Summary

HIV, the causative agent of AIDS, is a retrovirus and during viral assembly it must identify and capture its capped, polyadenylated genomic RNA from the background excess of cellular RNAs and encapsidate it specifically. Ours and others? previous studies have given us considerable insight into this process at a structural and mechanistic level. It is a critical stage in the virus life cycle involving highly conserved RNA and protein motifs and hence is susceptible to therapeutic intervention. Packaging inhibitors would also potentially generate an element of autovaccinatory immune response against the host?s repertoire of viral variants. Packaging involves interaction of the Gag polyprotein of the virus with a conserved RNA structure (the packaging signal - psi). It is becoming clear that a number of cellular chaperone molecules are involved and that the RNA must travel along a very specific route in the cell, probably involving the centrosome, to be encapsidated efficiently. We will investigate the HIV genomic RNA packaging process. We will use novel chemical structural biological methods including oligonucleotide structure probing by FRET and two colour coincidence detection, to analyse:- three dimensional psi RNA structures, the changes in RNA structure involved in capture and the stoichiometry of Gag interactions with the RNA packaging signal. These will be combined with classical 2D (biochemical probing) and 3D (crystallography, NMR) methods and structural predictive methodology. We will also study the process of RNA capture within the cell, the pathway along which the RNA must travel to be encapsidated and the cellular chaperones involved. We will probe the molecular interactions involved with subcellular fractionation and mass spectroscopy and with siRNA knockdown. We will use targeted oligonucleotides to probe these interactions and confirm their importance and to identify therapeutic targets. These studies will unravel a critical process in HIV assembly which has a potentially high therapeutic index for antiviral intervention. We will also translate the findings into better design of lentiviral vectors which should enhance their efficiency and lead to better clinical tools for diagnostic and therapeutic uses.

Publications

10 25 50
 
Description Milstein award
Amount £300,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 03/2009 
End 11/2012
 
Description Interdisciplinary links with Dept of Chemistry 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Identification of packaging signals in lentiviral RNA
Collaborator Contribution Collaboration has resulted in award of a Milstein grant from the MRC
Impact Milstein Grant from MRC
Start Year 2009