Defining the arenavirus interactome

Lab rotation project description

During the 7 week rotation, the student will express NP and Z proteins of the Lujo strain with a fluorescent tag in mammalian cells. The localisation of these tagged proteins will be analysed by immunofluorescence and the proteins purified from cells in preparation for mass spectrometry.  In parallel, NP and Z will be cloned into bacterial expression vectors and expression trials for purification and downstream structural analysis performed.

Week 1: Cell culture techniques and transfection

Week 2: Transfection and cloning

Week 3: Protein expression in mammalian cells,- detection by Immunofluorescence, western blotting

Week 4: Refinement of week 3

Week 5: Purification trials from mammalian cells, coomassie staining, data analysis and write up

Week 6: Expression of proteins in bacteria at different temperatures, coomassie staining

Week 7: Data compilation, analysis and write-up

Training will be provided in cell culture (sterile technique) and protein expression and purification, protein detection methods (immunofluorescence and western blotting) and data analysis. There will also be the opportunity to present the project at lab meetings and to the wider ‘One Virology’ group.

Fact file

Research theme

Molecules, cells and organisms

Location

School of Veterinary Medicine and Science

Rotation

Contact

Main (joint) supervisor:

Toshana Foster

toshana.foster@nottingham.ac.uk

2nd supervisor

2nd (joint) supervisor:

Janet Daly

janet.daly@nottingham.ac.uk


BBSRC Doctoral Training Partnerships
 

Linked PhD Project Outline

The recent Ebola epidemic highlights the immense impact human viral haemorrhagic fevers (VHFs) have on human health and on the socio-economic status of the developing world.  Arenaviruses are the largest family of VHF-causing viruses; they have worldwide distribution and are endemic in South America and West Africa, mainly in Sierra Leone, Liberia and Nigeria. 

The current outbreak by the most common arenavirus, Lassa, in Nigeria, makes it imperative to understand the molecular basis of viral pathogenesis and immune evasion, to identify factors that drive viral emergence and to identify drug targets. The Arenaviridae family is divided into Old World (OW - endemic in West Africa) and New World (NW - endemic in South America) viruses based on their phylogeny, geographical distribution and serological cross reactivity. Arenaviruses cause persistent infections in their natural rodent hosts and viral transmission to humans occurs through direct contact with infectious materials or exposure to rodent urine/faeces. Fatality rates are extremely high; there is no vaccine, the few therapeutic options are ineffective and diagnostic tools are poor. The overarching aim is to map the complex pathways that are responsible for potentiating and antagonising arenavirus infection and, importantly, explore the molecular basis of host adaptation. 

Scientific Question: What factors drive emergence and immunosuppression during arenavirus infection?
A distinctive feature of arenavirus infection in humans is the general immunosuppression that often leads to widespread, uncontrolled viral replication in various organs. Disease can also vary from asymptomatic to fatal implying that the interplay between the host immune response and viral replication is a major predictive factor for disease outcome. OW and NW viruses trigger differing immune responses; in OW infections, immunosuppression is observed whilst initial elevated cytokine levels, important in the early control of virus replication, are observed in NW infections, followed by a shift to a pro-inflammatory response. It remains to be discovered what drives the observed virus group specificity and what mechanisms govern the disparate responses to the host innate immune defence.The main orchestrators of viral replication and immunosuppression are viral proteins NP and Z.

The student will determine the similarities and differences in the host co-factors and restriction factors of OW and NW arenavirus NP and Z proteins, with particular focus on pathogenic versus non-pathogenic strains as the molecular basis for this differential virulence is unknown. The student will develop protein tagging methods and use minigenome systems, RNAi and proteomic screens for identification of novel interacting partners. Developing these methods should reveal novel restriction or resistance proteins and co-factors that define cellular tropism and cross-species transmission differences. Linked to this will be the use of structural methods to elucidate the molecular details of the interactions of NP and Z with host factors; exploiting these interactions is key in the application of this research to the design of therapeutics. This will involve cloning, expression and purification the viral proteins for structure determination of apo-structures or of protein complexes.

 
 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk