Dissecting the mechanism of action of CHD1L, a novel regulator of HIV-1 infection

Lead Research Organisation: University of Cambridge
Department Name: Medicine


HIV, the virus that causes AIDS, remains an important cause of ill health and mortality, with 37 million people living with the virus worldwide and annually being responsible for around 1 million deaths. This is despite significant progress in antiviral treatments. The virus interacts with infected human cells, and some of these interactions involve host defence molecules that critically affect the ability of the virus to complete its life cycle. This knowledge has informed the targeting of crucial viral processes in current treatment regimes. Most screening studies on how human genetics affect HIV replication have been conducted on European populations, despite more than 70 % of infected individuals and nearly half of new infections each year being in Africa. A recent study conducted by our collaboration has revealed a novel gene which has an enormous impact on HIV replication in patients of African ancestry. Upon infection, the level of the HIV in the body of the infected patient reaches a steady state and this level is a key factor in determining how quickly the immune system is compromised by the virus. This, in turn, is an important determinant of how long an HIV-infected individual takes to progress to AIDS. Our genomic study shows that a higher level of expression of a gene, chd1l, is associated with a lower steady state level of virus. The strength of the association between the level of protein produced by this gene CHD1L and the HIV level is equivalent to that of well described mutations in CCR5, one of the molecules necessary for most commonly transmitted viruses to gain entry into a cell, and these mutations are known to confer significant protection from HIV in patients harbouring them. Thus CHD1L has the potential to have an enormous impact on HIV replication.

It remains unclear, however, how CHD1L is able to inhibit HIV infection. Here we propose research to understand the interaction between CHD1L and HIV. We aim to pinpoint the step(s) in the virus life cycle at which CHD1L may be exerting an effect, through quantifying the markers of the different stages in cells with and without CHD1L. CHD1L unwinds the packaging of DNA in response to specific signals in the cell. There are two candidate parts of the life cycle where this could be important. 1. In order to express its proteins, HIV inserts its genome into the host DNA in a process called integration. CHD1L could affect the efficiency and/or specificity of this process. 2. DNA also needs to be unwound for the encoded proteins to be expressed; again CHD1L could affect this process.

We will also determine whether CHD1L binds to any specific HIV proteins by using highly sensitive techniques to see what CHD1L is bound to in infected and uninfected cells. In this way, we will identify the proteins and pathways in the cell through which CHD1L affects HIV replication. We will also identify features of the CHD1L protein that are essential for its ability to limit HIV infection by making changes to key parts of the protein and monitoring the ability of modified versions of the protein to inhibit HIV infection. Interactions with specific viral or cellular proteins could be exploited therapeutically to generate new treatments for HIV. The knowledge generated by our proposed experiments is necessary to translate our initial observation into clinically useful treatment for this devastating virus. The group of experts assembled to carry out this work are uniquely well-placed position to do this due to their expertise in the technology required for this work and their links to clinical and commercial partners.

Technical Summary

HIV remains an important cause of morbidity and mortality. We have uncovered a novel polymorphism in African populations correlated with HIV viral load set point. We have shown that the gene responsible is CHD1L, is a novel cellular factor negatively regulating HIV-1 infection. To study its mechanism of action, we have knocked out CHD1L in inducible pluripotent stem cell (iPSC)-derived macrophages. We have also created CHD1L knockdown vectors and expression vectors and are validating them in Jurkat and THP-1 cells. We will discern cellular pathways involved in CHD1L's regulation of HIV-1 with these cells, using transcriptomic and TMT-mass spectrometry based proteomic approaches. To identify protein binding partners of CHD1L, we will conduct SILAC-IP using lysates from uninfected and HIV infected cells. We will pinpoint the step(s) of the virus life cycle affected by CHD1L using engineered vectors expressing different fluorophores upon integration and upon activation of the viral promoter. This will be complemented by qPCR based techniques to detect the HIV genome in different stages of its life cycle, direct visualisation of the chromosomal location of viral integration sites with FISH, and assessment of nucleosome remodelling of the viral promoter with chromatin histone immunoprecipitation assays.

The results of the above studies will be followed up with specifically designed experiments. Effects on viral integration will be confirmed with in vitro integration assays. Protein interactions will be confirmed with traditional immunoprecipitation/western blot techniques, using relevant mutant viruses and expression vectors. Should CHD1L alter the viral integration landscape, the distribution of the integration site will be determined. We will also create a panel of mutant CHD1L expressors, inactivating different domains/residues of the protein to examine the specific biochemical activities of CHD1L that are crucial for its regulation of HIV.

Planned Impact

Work at the cell/virus interface has the potential to reveal cellular factors that interact with a virus. For a global pathogen such as HIV, this is important as it can unveil new therapeutic targets to be exploited in the ongoing fight to suppress and eradicate the virus. Cellular factors that interact with HIV place a constraint on the virus's ability to mutate the sequence encoding the interacting viral factor, and provide a possible route to highly efficacious treatments with a higher threshold for mutational escape than therapeutic approaches targeting purely virus specific factors and processes. The host factor we are studying, which exerts as huge an impact on the set point viral load as mutations in CCR5, is likely to significantly impact on the replication of HIV. We have a track record of developing new paradigms of antiviral therapy for HIV. Once we have generated the knowledge necessary through this proposal, we hope to extend this further and identify processes suitable for high throughput screening of chemical entities as novel drugs, focussing on these pathways. Our research will be of interest to the pharmaceutical industry, both at the smaller biotech level and larger pharmaceutical companies. Ultimately the beneficiaries are patients with HIV infection and the healthcare budget.

Academic impacts are vital. We have, with previous MRC funding, developed novel techniques for RNA structural analysis and for probing the structural changes and sites of interaction when RNA and protein bind to each other. Both of these techniques provide new tools for researchers in many closely and more distantly related fields. This research, that covers so many basic areas of cell biology and virology will undoubtedly lead to new knowledge on disease processes (many of which, such as cancer, involve DNA repair cellular factors) and will lead ultimately to quality of life enhancement. The current proposal has the potential to create advances in understanding mechanisms of viral infection and the control of cell division and how they malfunction in cancer, which will impact widely.

In the longer term our work may contribute to wealth creation and economic prosperity, dependent on our ability to translate this research into practical therapeutic targets. As part of the national CHERUB Collaborative, we have established links with a network of investigators, including clinical triallists, and are thus in a position to readily translate any exploitable outcomes into clinical studies.

Whilst not directly contributing to increased public awareness in itself, the appointed researchers and the PIs will take every opportunity to present our research, and the importance of basic research, which leads to translational outputs to the general public, and to increase the general understanding of science. We aim to attract R&D investment to commercialise any outputs. The project management, bioinformatic and IT skills required to perform this work enhances the ability of individuals employed on the project to work in both academic and, if they wish, non-academic professions.

Timescales for these benefits are difficult to estimate but for basic research to translate into therapeutic benefit is likely to take at least 5-10 years.


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