Regulation of clonality in a natural retrovirus infection

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.

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.

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.