PhD project proposal template 2012-13
Principal Supervisor name and department:
Dr Emma Anderson, School of Life Sciences, University of Warwick
Second Supervisor name and department:
Martin Bushell (Leicester)
Where will the student be based?
Warwick
PhD project title:
Investigation of the mechanism of -1 frameshifting on HIV-2 RNA
Project description:
Ribosomal frameshifting is a mechanism by which a messenger RNA (mRNA) can encode more than
one protein, and is utilized by many viruses to expand their proteome. A frameshift signal in the
mRNA causes translating ribosomes to pause and then slip either backwards or forwards into a
different reading frame, before continuing to translate an alternative protein. In human
immunodeficiency virus (HIV), ribosomal frameshifting allows the production of the structural
proteins and the enzymes of the virus from the same mRNA, in the correct ratio for efficient
replication. In the case of HIV-1, this process has been reasonably well-characterised, with a
frameshifting efficiency of 5-10% being optimal for viral replication. Frameshifting in HIV-2 has not
been investigated, and had been assumed to occur in the same way as for HIV-1. However,
preliminary data from our laboratory suggests that the efficiency of frameshifting for HIV-2 is much
higher. This project will investigate the mechanism by which ribosomal frameshifting occurs on HIV-2
mRNA.
Sequence data is available for many strains of HIV-2 and the very closely related SIVmac. Bioinformatic
analysis, including covariance analysis, of the sequence around the site of frameshifting will be
undertaken to predict the RNA secondary structure of the frameshift signal and the predicted
structure will then be tested using RNA structure mapping. The effect of mutations predicted to
disrupt the structure will be tested in in vitro translation assays and in viral replication assays in cells.
The effect of both cellular and viral proteins on the frameshifting efficiency will be tested in in vitro
translation assays using recombinant proteins and cell lysates, and the binding site of any identified
proteins mapped.
Finally, the effect of the rate of translation on the frameshifting efficiency will be investigated – does
the speed with which ribosomes encounter the frameshift signal determine their likelihood of
frameshifting?
The aim of these studies is to determine what factors affect the rate of frameshifting in mammalian
cells; why is the frameshifting efficiency of HIV-2 different to that of HIV-1?
Key experimental skills involved:
Bioinformatic analysis, RNA secondary structure prediction and mapping.
Molecular biology, mutagenesis, protein expression and purification.
Tissue culture and viral replication assays.
PhD project proposal template 2012-13
References:
Characterization of the frameshift stimulatory signal controlling a programmed -­­1 ribosomal
frameshift in the human immunodeficiency virus type 1. Dulude D, Baril M, Brakier-­­Gingras L.
(2002) Nucleic Acids Research 30: 5094-­­5102.
Contact details for application enquiries: Dr Emma Anderson
e.c.anderson@warwick.ac.uk
Keywords:
Translation; frameshifting; virus.
PhD project proposal template 2012-13
Please state below which hazards may be connected with this studentship:
The studentship will entail work involving:
√ if applies
Chemicals – high toxicity and category 1 or 2 substances
Organo-phosphate or carbamate pesticides
Skin or respiratory sensitising agents (e.g. insect parts, organic
dusts from animals, spores, pollen, antibiotics, fibres, chemical
sensitisers, wood dust etc)
Radionuclides
Significant manual handling
Mechanical repetition where the frequency and duration are
significant
Working in areas where there are temperature extremes
Driving vehicles (tractors, fork lifts, ATVs etc)
Crop planting, harvesting, recording or grading
Working in close proximity to bees or other stinging insects
Working at height [>2 m] (using various types of access
equipment)
Working with noisy or vibrating equipment
Working at night (between 11.00pm and 6.00am)
Food handling
Other significant hazards (specify)
X