Mechanisms within the ESCRT machinery required for HIV-1 budding

Lead Research Organisation: King's College London
Department Name: Immunology Infection and Inflam Diseases

Abstract

Production of infectious Human Immunodeficiency Virus-1 (HIV-1) requires the separation of the nascent viral particle from the infected cell at the end of the assembly process. This late event in HIV-1 replication requires the resolution of a membrane tether by specialized cellular proteins, namely the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Importantly, there is a growing list of pathogenic viruses that also require the ESCRT machinery for replication, including Ebola virus, Hepatitis B virus, Herpes Simplex virus type 1, human T-cell leukemia virus type 1 (HTLV-1), Lassa virus and Parainfluenza virus 5. Hence, the pharmacological inhibition of the ESCRT machinery may offer a unique therapeutic opportunity to treat a broad range of pathogenic viral infections. However, there are obvious toxicity mechanisms for future inhibitors of the ESCRT pathway as it is primarily involved in essential aspects of cell biology, including the resolution of a membrane tether that connects the daughter cells at the final stage of cell division. The general aim of this research grant is to delineate specific mechanisms within the ESCRT machinery that are required for HIV-1 replication as opposed to this machinery?s role in cellular functions. These studies are needed to understand essential mechanisms of HIV-1 replication and cell biology and they will also contribute to determine the therapeutic potential of the ESCRT-machinery.

Technical Summary

The last event in the assembly of infectious Human Immunodeficiency Virus (HIV-1) requires a membrane scission event that separates the nascent viral particle from the infected cell. This process is mediated by a late budding activity (L-domain) encoded by a short aminoacid motif (P/SAP) in the structural viral protein gag. It is now established that the HIV-1 L-domain facilitates egress via the recruitment of the Endosomal Complex Required for Transport (ESCRT) machinery. Moreover, there is a growing list of pathogenic human enveloped viruses that also require the ESCRT machinery for replication, namely Ebola virus, Hepatitis B virus, Herpes Simplex Virus type 1, HTLV-1, Lassa virus and Parainfluenza Virus 5. Consequently, the pharmacologic inhibition of the ESCRT machinery may offer a therapeutic opportunity against a broad range of enveloped viruses, but the fact that the ESCRT complexes are primarily involved in essential aspects of cell biology such as endosomal sorting into the Multivesicular Bodies and the resolution of the midbody at the end of cell division raises obvious potential mechanisms of toxicity for future inhibitors of the ESCRT machinery. Therefore, understanding the mechanisms underpinning the functional complexity within the ESCRT machinery and its regulation is not only needed to determine the therapeutic potential of this pathway, but also to further illuminate basic aspects of HIV-1 pathogenesis and cell biology. Thus, the general aim of this research grant application is to define the specific mechanisms within the ESCRT machinery that are required for HIV-1 budding as opposed to the role of this machinery in cytokinesis and endosomal sorting.

Publications

10 25 50
 
Description MRC Research Grant G0802777
Amount £967,648 (GBP)
Funding ID G0802777 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2011 
End 07/2015
 
Description Wellcome Trust Project Grant (Uncovering the role of MITD1 in ESCRT function)
Amount £259,231 (GBP)
Funding ID WT093056 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2011 
End 12/2013
 
Description Wellcome Trust Senior Investigator Award
Amount £1,299,251 (GBP)
Funding ID WT102871MA 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2014 
End 06/2019
 
Title Hela YFP-Tsg101, Hela Cherry-Cep55, Hela YFP-ALIX 
Description These are stable cell lines expressing a fluorescent versions of several components of the ESCRT machinery and other interacting proteins 
Type Of Material Cell line 
Year Produced 2007 
Provided To Others? Yes  
Impact These cell lines have been shared with several researchers that are investigating the role of cellular proteins in HIV-1 replication and cytokinesis. 
 
Title Plasmids encoding several variations of the ESCRT subunits 
Description Our laboratory has created many plasmids encoding fusions of the ESCRT subunits to various tags, such as fluorescent proteins, GST, yeast two hybrid, etc 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact We constantly share these plasmids on request with many research groups that work in several areas of research, such as virology and cell biology 
 
Title Plasmids encoding several variations of the ESCRT subunits 
Description Our laboratory has created many plasmids encoding fusions of the ESCRT subunits to various tags, such as fluorescent proteins, GST, yeast two hybrid, etc 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact We constantly share these plasmids on request with many research groups that work in several areas of research, such as virology and cell biology 
 
Description Collaboration with Professor Wes Sundquist 
Organisation University of Utah
Country United States 
Sector Academic/University 
PI Contribution Identification and functional characterization of ULK3 as a kinase that regulates cytokinesis via the phosphorylation of ESCRT-III. We identified ULK3 as an ESCRT-associated kinase involved in the regulation of cytokinetic abscission. We established functional assays to determined the relevance of ESCRT-III phosphorylation in abscission regulation
Collaborator Contribution Biochemical and structural characterization of ULK3. Specifically, our partners determined the structure of ULK3, and how this kinase interacts with ESCRT-III. Further biochemical characterization identified ULK3 phosphorylation sites in ESCRT-III.
Impact Caballe A, Wenzel DM, Agromayor M, Alam SL, Skalicky JJ, Kloc M, Carlton JG, Labrador L, Sundquist WI*, Martin-Serrano J*. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins. Elife. 2015 May 26;4:e06547.
Start Year 2013
 
Description Collaboration with Roger Williams at the MRC LMB 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Functional characterization of UBAP1 as a novel subunit of ESCRT-I
Collaborator Contribution Structural and biochemical characterization of UBAP1 as a novel subunit of ESCRT-I
Impact Agromayor M, Soler N, Caballe A, Kueck T, Freund SM, Allen MD, Bycroft M, Perisic O, Ye Y, McDonald B, Scheel H, Hofmann K, Neil SJ, Martin-Serrano J*, Williams RL*. The UBAP1 subunit of ESCRT-I interacts with ubiquitin via a SOUBA domain. Structure. 2012 Mar 7;20(3):414-28
Start Year 2011
 
Description Collaboration with Roger Williams at the MRC LMB 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Functional characterization of MITD1 as a new component of the ESCRT pathway
Collaborator Contribution Structural and biochemical characterization of MITD1
Impact Hadders MA, Agromayor M, O Takayuki, Perisic O, Caballe A, Kloc M, Lamers MH, Williams RL, Martin-Serrano J*. The ESCRT-III binding protein MITD1 is involved in cytokinesis and has an unanticipated PLD-fold that binds membranes. Proc Natl Acad Sci U S A. 2012, Oct 23; 109 (43):17424-9.
Start Year 2011