Regulation of clonality in a natural retrovirus infection

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

Context

A retrovirus is a virus that integrates its DNA into the genome of the cell that it infects, where the virus can lie dormant or "latent" indefinitely. This retroviral latency is the greatest single obstacle to the eradication of a retroviral infection: although drug treatment for HIV infection is highly efficient at suppressing HIV replication, the virus is not cleared from the body because there is a reservoir of cells containing latent HIV, from which the virus reactivates if drug treatment is stopped, resulting in the development of AIDS. Understanding retroviral latency is also of critical important in the development of safe retrovirus-based viruses used in gene therapy.

Human T lymphotropic virus type 1 (HTLV-1) is the other main disease-causing retrovirus in humans. Present mainly in the tropics, HTLV-1 causes 2 types of disease: an aggressive leukaemia, which is almost uniformly fatal, and a chronic disabling paralytic disease, which is also untreatable. There is no vaccine.

Retroviral latency has been difficult to study in HIV infection, because HIV rapidly kills the infected cell when the virus is reactivated. HTLV-1, in contrast, does not kill the infected cell but drives it to proliferate.

Aims

The aim of this programme is to identify how retroviral latency is regulated, to allow the virus to persist despite the host immune response and drug treatment. We have developed a sensitive, high-throughput technique to map the integration site of retroviruses in the host DNA and, crucially, to quantify accurately the abundance of each clone of infected cells. We have shown that the integration site of the retrovirus, which is different in each clone of infected cells, is a major factor that determines the abundance of that clone. In Part A of the proposed programme, we will use this new technique to identify the features of the integration site that are associated with reactivation of the virus and proliferation of the cell. To do this we will study DNA from peripheral blood taken from patients with the different diseases caused by HTLV-1. In Part B of the programme we will test specific hypotheses on the molecular mechanisms of retroviral latency: 2 hypotheses that we have already formulated, and further hypotheses that we develop from the data obtained in Part A.

Applications and benefits

The benefits of this work will be two-fold. First, it will lead to fundamental advances in the understanding of the regulation of latency of retrovirus such as HIV and HTLV-1, and viruses used in gene therapy. Second, the ability to predict and explain why certain individuals develop serious diseases from HTLV-1 infection whereas others remain healthy will provide tools for prognosis and monitoring response to treatment of the HTLV-1-associated leukaemia and the paralytic disease. The mathematical techniques developed will be of wide application in medicine, ecology and population biology.

Technical Summary

Objectives

i) To identify and quantify the genetic and epigenetic factors that determine the number, abundance and spontaneous proviral expression of HTLV-1-infected T cell clones in asymptomatic carriers and patients with HTLV-1 diseases;

ii) To test mechanistic hypotheses on the regulation of HTLV-1 proviral latency in naturally-infected T cells.

Methods & experimental approach

i) Materials: genomic DNA from primary peripheral blood mononuclear cells from subjects with HTLV-1 infection.

Methods: High-throughput mapping and quantification of proviral integration sites in the genome (350 gDNA samples per Illumina HiSeq 2000 flow-cell). Laboratory protocols, work flow and sequences of primers and linkers are given in Gillet et al 2011. Bioinformatic, mathematical and statistical techniques are outlined in Gillet et al 2011 and extended in Berry et al 2012 (Bioinformatics 28, 755-762).

ii) Materials: Fresh primary PBMCs from HTLV-1+ subjects; HTLV-1+ CD4+ T cell clones isolated from PBMCs by limiting dilution; HTLV-1+ T cell lines.

Methods:
Chromatin immunoprecipitation (ChIP) to localize and quantify binding to chromatin of host proteins involved in regulating gene expression (RNA Pol II, cohesin etc) and epigenetic markers (H3K9Me3 etc). Electrophoretic mobility shift assay (EMSA) to confirm binding of proteins to proviral DNA.
Chromosome conformation capture (3C) to detect and map chromatin loops formed between proviral DNA and host chromatin.
Flow-sorting of cells according to stage of cell cycle.

iii) Generalized linear models; logistic regression; ordinary differential equations; standard parametric & non-parametric statistics.


Application & exploitation

We are developing a rapid, semi-quantitative assay of clonality for use in clinical management of ATLL.

Planned Impact

In addition to the academic beneficiaries, four groups will benefit from this research, in the following ways:

1) Clinicians involved in the management of individuals with HTLV-1 infection and patients with HTLV-1 infection and the associated diseases. They will benefit through the use of assays to assist in the diagnosis, prognosis and response to therapy of HTLV-1 diseases, especially ATLL. Treatment for ATLL remains highly unsatisfactory, and there is active research involving clinical trials of novel therapeutic combinations (AZT, IFN-alpha, arsenic trioxide etc). The ability to predict who is at risk of developing ATLL and to follow the response to therapy accuarately and in real time will constitute a major advance in the management of ATLL.

2) Clinicians and patients involved in gene therapy. Gene therapy is also at an early stage of development, and there is a strong need for improved methods to identify safe genomic harbours for therapeutic gene insertion and to monitor the results of gene therapy. Several patients have developed leukaemia following retroviral gene therapy for X-linked SCID. The previous methods available for detecting individual clones carrying a given retroviral insertion were unable to quantify clone abundance accurately, and our methods represent a significant advance in this important developing application.

3) Patients with other retroviral infections, including notably HIV-1, will benefit from the increased understanding of the mechanisms of retroviral latency. In addition, patients with HTLV-1-infection will benefit through improved detection, diagnosis, prognosis and treatment of the malignant and inflammatory diseases associated with HTLV-1 infection.

4) Staff working on the project will develop skills in a set of laboratory and analytical techniques that are in strong - and increasing - demand. In particular, the combination of the novel mathematical techniques and the bioinformatic analysis will find wide application in highly active fields of research. This will put the staff members in a strong position to advance their careers.

Publications

10 25 50
 
Description The human T-cell leukaemia virus HTLV-1: transcriptional heterogeneity at the single-cell level
Amount £76,221 (GBP)
Funding ID MR_T029005_1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2020 
End 12/2022
 
Description Wellcome Trust Investigator Award
Amount £1,927,877 (GBP)
Funding ID 207477/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2018 
End 07/2022
 
Title Protocol for high-throughput mapping and quantification of retroviral integration sites 
Description A laboratory protocol and guide to the data analysis for the mapping and quantification of retroviral integration sites in the host genome. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2017 
Provided To Others? Yes  
Impact The protocol has been widely adopted, notably in the field of HIV-1 infection, where it has changed the understanding of the latent reservoir of HIV-1 that persists under antiretroviral drug treatment. 
 
Title Protocol for quantification of diversity (number) of integration sites and T-cell receptors 
Description Mathematical approach to estimation of population diversity. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2015 
Provided To Others? Yes  
Impact The approach described has been adopted by both immunologists, to estimate T-cell receptor diversity, and by virologists, notably in HIV-1 infection. 
URL https://cran.r-project.org/web/packages/DivE/index.html
 
Title Retroviral integration sites: high-throughput mapping and quantification 
Description We described a novel protocol for the laboratory, and an accompanying bioinformatics method, to map and quantify retroviral integration sites. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact The use of our protocol, published in 2011 (Gillet al 2011: Blood 117, 3113-3122), has made a major impact in the analysis of integration sites in HIV-1 infection and other retroviral infections. 
 
Description Impact of HTLV-1 infection on host genome 
Organisation University of Kumamoto
Country Japan 
Sector Academic/University 
PI Contribution I have been appointed to the Scientific Advisory Board of the International Research Center for Medical Sciences in Kumamoto. I take part in the regular review of the research of the Institute. One of my recent post-docs has now been appointed as a member of staff in the Institute; we maintain a close collaboration and exchange visits both Japan-UK and UK-Japan.
Collaborator Contribution My collaborating partner (ex-postdoc) is a) continuing collaborative experimental work in the laboratory and b) helping us to negotiate the collection of valuable samples from other centres in Japan for our research.
Impact The first manuscript of a scientific paper from this collaboration is now in preparation.
Start Year 2013
 
Description Impact of bronchiectasis and HBV on HTLV-1 clonality 
Organisation University of Adelaide
Country Australia 
Sector Academic/University 
PI Contribution We are analysing samples of clinical material provided by our collaborator; we anticipate that this will culminate in at least one original scientific publication.
Collaborator Contribution Our partner is raising further research funds in Australia, collecting the essential clinical samples, and contributing to the design and interpretation of the experiments.
Impact Scientific publications are anticipated, but this collaboration is still in its early stages.
Start Year 2013
 
Title DivE 
Description An online R package for the calculation of total population diversity of complex populations. The publication describing this application is Laydon et al 2014: PLoS Comput Biol 10(6): e1003646; this publication has also been linked to this award. 
Type Of Technology Software 
Year Produced 2014 
Impact The method and the software have been adopted by research groups both in immunology and virology. 
URL http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1003646
 
Description Audiobook of Scientia article - HTLV-1 - the forgotten cousin of HIV 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact An audiobook version of my article for the public communication of science company, Scientia.
Year(s) Of Engagement Activity 2018
URL http://www.scipod.global/htlv-1-the-forgotten-cousin-of-hiv-professor-charles-bangham-imperial-colle...
 
Description Imperial College podcast on PNAS paper (Satou et al 2016) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A podcast for Imperial College was made, to disseminate the results that we published in our research on the human T-cell leukaemia virus, HTLV-1.
Year(s) Of Engagement Activity 2018
URL https://wwwf.imperial.ac.uk/imedia/content/view/6190/the-virus-that-causes-cancer/
 
Description Scientia article: HTLV-1 - the forgotten cousin of HIV 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A magazine article written, in consultation with me, by an organization whose objective is the public communication of science - Scientia. An audiobook version of this article was also produced.
Year(s) Of Engagement Activity 2018
URL https://www.scientia.global/wp-content/uploads/Charles_Bangham/Charles_Bangham.pdf