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Mechanisms of dynein-mediated transport of Marek’s Disease Virus


Warwick Medical School

Sunday, January 10, 2021 Competition Funded PhD Project (Students Worldwide)

About the Project

This project is available through the MIBTP programme on a competition basis. The successful applicant will join the MIBTP cohort and will take part in all of the training offered by the programme. For further details please visit the MIBTP website - https://warwick.ac.uk/fac/cross_fac/mibtp/

The sustainability of global food production is under threat from the increase in human population, reduced availability of natural resources and climate change. With a current global production of 55 billion chickens annually, poultry meat and eggs dominate animal protein products for human consumption world-wide. The UK poultry industry, contributing around £3.5 billion to the economy annually, provides more than 90% of the poultry meat and egg for the country. Avian diseases, particularly those induced by viral pathogens, remain a major threat for sustainable poultry production. Marek's disease is one of the most widespread and economically important diseases of poultry, with global losses estimated to more than US $2,000 million. MD is a fatal, highly contagious, neoplastic disease affecting chickens. It is caused by an alphaherpesvirus called Gallid herpesvirus-2 or Marek's Disease Virus (MDV). Advancing our understanding of MDV by identifying and characterising virus-host interactions will enable us to develop interventions that limit the transmission of MDV. MDV is a large DNA virus that is strictly cell-associated. Thus live infected cells have to be used to maintain the infectivity of the virus and it is highly dependent on the host cytoskeleton to transport MDV from entry site to nucleus, and new virus particles via Golgi back to the cell surface to transmit to neighbouring cells.

The major minus-end directed motor dynein is essential for transporting viruses from cellular entry point to the nucleus where they replicate. The most likely candidate proteins to mediate the linkage of the virus to the intracellular transport machinery are the capsid-associated inner tegument proteins UL36 and UL37. However, the molecular detail of how the complex of UL36-UL37 mediates loading onto dynein isn’t known for any herpesvirus. Here we aim to identify host proteins that interact with MDV UL36-UL37 and could mediate the interaction with the dynein motor, we will test physiological importance of candidates by imaging MDV capsid motility in chicken cells depleted for those host proteins, map interaction surfaces and will attempt to reconstitute UL36-UL37 transport by dynein in vitro.

Thus the project will combine proximity-dependent biotinylation and mass spectrometry identification, live cell imaging using lattice light sheet microscopy, quantitative image analysis and particle tracking, protein purification and imaging-based in vitro reconstitution assays.

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Animal Health and Welfare. Understanding the Rules of Life: Microbiology

Techniques that will be undertaken during the project:
Animal cell culture; molecular biology to clone cDNAs and introduce deletions and point mutations into expression constructs; RNAi and DNA transfection to deplete proteins of choice and supplement with wild-type and mutant versions; proteomics to identify interacting proteins; protein purification and protein-protein interaction analysis; live cell imaging assays (e.g. imaging infected chicken cells using lattice light sheet and spinning disk confocal microscopy); total internal reflection fluorescence microscopy paired with biochemical reconstitution assays; image and statistical data analysis using ImageJ and Matlab.

References

Luxton GW, Lee JI, Haverlock-Moyns S, Schober JM, Smith GA. The pseudorabies virus VP1/2 tegument protein is required for intracellular capsid transport. J Virol. 2006 Jan;80(1):201-9. doi: 10.1128/JVI.80.1.201-209.2006. PMID: 16352544; PMCID: PMC1317523.

Zaichick SV, Bohannon KP, Hughes A, Sollars PJ, Pickard GE, Smith GA. The herpesvirus VP1/2 protein is an effector of dynein-mediated capsid transport and neuroinvasion. Cell Host Microbe. 2013 Feb 13;13(2):193-203. doi: 10.1016/j.chom.2013.01.009. PMID: 23414759; PMCID: PMC3808164.

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