Role of the secretory pathway in HIV-1 egress from T cells

Lead Research Organisation: University College London
Department Name: Infection

Abstract

The human immunodeficiency virus type-1 (HIV-1), the cause of the global AIDS pandemic, replicates primarily in cells of the immune system called CD4+ T cells. One of the most important steps in the virus life cycle is constructing new virus particles that are released by infected cells, transmitting infection to neighbouring cells. Viral assembly is carefully controlled to ensure that the necessary components are transported to the correct location in the cell at the right time. To do this, HIV-1 hijacks cellular machinery and transport routes. Understanding how this machinery works normally, and how HIV-1 interacts with it is the aim of this project. Information from experiments performed with other cell types has provided clues about which molecules may be important, and these will be tested to see if they control the flow of HIV-1 traffic in CD4+ T cells. There is evidence that HIV-1 infected cells receive signals from uninfected cells that trigger the assembly and release of HIV-1, helping viral spread. Experiments designed to identify these signals will also be performed. Studying HIV-1 assembly and spread is essential for better understanding how it causes disease, and will facilitate design of new anti-HIV-1 drugs.

Technical Summary

The aim of this project is to establish the role of the secretory pathway in HIV-1 egress from T cells. Evidence from model, non-lymphoid systems indicates that HIV-1 hijacks elements of secretory pathways to transport viral proteins and coordinate virus assembly, but almost nothing is known about HIV-1 assembly and egress in primary CD4+ T lymphocytes. Haematopoietic cells are unusual because in addition to the ubiquitous constitutive secretory pathway, they contain specialised organelles transported via regulated secretion in response to stimuli such as cell-cell contact. A regulated secretory pathway exists in CD4+ T cells, but little is known about the cellular compartments involved, or the triggers for activation of secretion. By contrast, molecules involved in regulated secretion by CD8+ T cells at the immune synapse are well characterised and may provide clues about factors mediating HIV-1 assembly in CD4+ T cells. The key goals of this project are to:
i) broadly characterise how CD4+ T cells traffic cellular and viral proteins via constitutive and regulated secretory pathways;
ii) identify cellular and viral molecules controlling regulated secretion in CD4+ T cells;
iii) evaluate the contribution of CD4+ T cell secretory pathways to viral dissemination between T cells, and to investigate strategies of inhibition.
Experiments designed to dissect the secretory pathway in CD4+ T cells and its subversion by HIV-1 will be performed using cells from normal individuals, and from individuals with defined genetic defects in molecules that control the transport and release of secretory lysosomes from CD8+ T cells. Immunofluorescence confocal microscopy and electron microscopy, subcellular fractionation and biochemical analysis will be employed to colocalise HIV-1 with molecules that undergo regulated secretion from CD4+ T cells. Emphasis will be placed on transport of the viral glycoprotein Env, and characterisation of associated compartments, as less is known about Env trafficking and Env incorporation is absolutely required for viral infectivity. The cellular cytoskeleton is intimately linked with secretory pathways, immune cell interactions and is implicated in HIV-1 egress and spread. To identify novel proteins involved in the intracellular transport of Env and Gag and virus assembly a siRNA screen will be performed targeting kinesin, dynein and myosin molecular motors. By identifying cellular factors that regulate HIV-1 egress and dissemination we aim to achieve a greater understanding of HIV pathogenesis that should open up opportunities for design of novel therapeutic agents to treat HIV/AIDS.

Publications

10 25 50
 
Title Cover image 
Description Cover image for Journal of Virology 
Type Of Art Image 
Year Produced 2014 
Impact Raises profile of research publication 
 
Description Gates Foundation Group meeting
Geographic Reach North America 
Policy Influence Type Participation in advisory committee
 
Description MRC Project Grant
Amount £486,000 (GBP)
Funding ID MR/J008184/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2012 
End 09/2015
 
Description NIHR CBRC Capital Grant
Amount £150,000 (GBP)
Organisation National Institute for Health Research 
Department UCLH/UCL Biomedical Research Centre
Sector Public
Country United Kingdom
Start 01/2012 
End 01/2012
 
Description Travel grant
Amount £379 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2009 
End 02/2009
 
Description ADAP during cell-cell spread 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution collaborated on research an co-authored a publication
Collaborator Contribution collaborated on research an co-authored a publication
Impact publication in the journal Retrovirology
Start Year 2011
 
Description Cell to cell transmission of HIV and tetherin 
Organisation King's College London
Department School of Medicine KCL
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed experiments to generate data, analysed the data and wrote a paper reporting our findings that was published in the Journal of Virology
Collaborator Contribution A publication has arisen from this work.
Impact Work arising from this collaboration has been published in the Journal of Virology
Start Year 2009
 
Description Role of transport in 3 in HIV infection 
Organisation University College London
Department Division of Infection and Immunity
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed immunofluorescence experiments to localise HIV proteins in transportin 3 knockdown cells using wild type and mutant virus
Collaborator Contribution The research arising from this collaboration was published in PLoS Pathogens.
Impact The research arising from this collaboration was published in the peer reviewed journal PLos Pathogens
Start Year 2010
 
Description Weiss 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborated on project and co-authored publication
Collaborator Contribution Collaborated on project and co-authored publication
Impact Co-authored publication in Retrovirology
Start Year 2012
 
Description modeling 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Initiated project, collaborated on project and co-authored manuscript.
Collaborator Contribution Collaborated on project, performed analysis, provided data and co-authored manuscript.
Impact manuscript submitted
Start Year 2013
 
Description modeling 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Initiated project, collaborated on project and co-authored manuscript.
Collaborator Contribution Collaborated on project, performed analysis, provided data and co-authored manuscript.
Impact manuscript submitted
Start Year 2013
 
Description modeling 
Organisation University College London Hospitals NHS Foundation Trust
Country United Kingdom 
Sector Public 
PI Contribution Initiated project, collaborated on project and co-authored manuscript.
Collaborator Contribution Collaborated on project, performed analysis, provided data and co-authored manuscript.
Impact manuscript submitted
Start Year 2013
 
Description modeling 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Initiated project, collaborated on project and co-authored manuscript.
Collaborator Contribution Collaborated on project, performed analysis, provided data and co-authored manuscript.
Impact manuscript submitted
Start Year 2013
 
Title mathematical modelling 
Description online tool to model HIV spread during infection, interventions and disease progression 
Type Of Technology Software 
Year Produced 2015 
Open Source License? Yes  
Impact Publication. 
URL https://github.com/mleoking/leotaskapp
 
Description Hosted school work experience students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Two students from Clapton Girls' School Camden London undertook work experience in my laboratory

Positive feedback from school and requests for student visits
Year(s) Of Engagement Activity 2012
 
Description MRC Centenary 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We manned a stall helping children make viruses out of plasticine and performing calculations for experiments.
Year(s) Of Engagement Activity 2014
 
Description Media 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release and media interviews (Guardian, Daily Mail, The India Times) regarding our modeling paper published that showed HIV spreads like a computer worm.
Year(s) Of Engagement Activity 2015