Viral and cellular regulation of T-cell amino acid metabolism

Approximately 100 years since it was first transmitted to humans, the Human Immunodeficiency Virus (HIV) now infects almost 40 million people worldwide, and causes more than 1 million AIDS-related deaths every year. It is therefore critical to understand how HIV has been able to replicate and spread, and why HIV infection causes AIDS. "Proteomics" is the large-scale study of "proteins", the critical building blocks of living cells and organisms. During my PhD, I used proteomics to measure changes in the number and quantity of proteins at the surface of cells infected with HIV, and found that >100 proteins were specifically depleted by the virus. Proteins are themselves made up of long chains of "amino acids", and many of the proteins I found to be depleted by HIV are involved in transporting amino acids into cells. These amino acid "transporters" are relatively understudied, and may be attractive candidates for new therapies. I therefore wish to understand why amino acid transporters are targeted by HIV, and the importance of these transporters for "T-cells", the main cells of the immune system infected by HIV and progressively destroyed in patients with AIDS.

Amongst the HIV targets I identified were proteins called SNAT1 and SERINC3/5. I discovered that SNAT1 transports an amino acid called alanine into cells, and that an abundant supply of alanine is essential for normal T-cell function. Likewise, SERINC proteins are thought to incorporate an amino acid called serine into cell "membranes", which surround cells and separate their interiors into compartments. In the first part of my project, I therefore wish to determine why alanine is important for T-cells, and investigate the role of SERINC3/5 in T-cell serine incorporation. "Metabolism" refers to the chemical reactions which take place in living cells and organisms, and a number of these reactions involve amino acids. In other settings, changes in T-cell metabolism are known to be important in regulating T-cell function. In the second part of my project, I therefore wish to investigate in more general terms which metabolic reactions are altered in HIV-infected cells, and understand how these changes benefit the virus. Finally, in the third part of my project, I wish to combine my skills in proteomics with a new technology called "CRISPR" to identify amino acid transporters and metabolic reactions which are critical for normal T-cell function, even in the absence of HIV infection. Taken together, these data will be a valuable resource for other researchers in the field and will, I hope, lead to the development of new treatments for patients targeting amino acid transporters in T-cells.

I previously used TMT-based plasma membrane proteomics to gain a comprehensive, unbiased overview of cell surface proteins regulated by HIV infection, and discovered HIV-mediated downregulation of >100 novel plasma membrane protein targets. Amongst these, the amino acid transporter SNAT1 was downregulated by the HIV accessory protein Vpu, and the putative serine carriers SERINC3/5 were downregulated by Nef. I identified alanine as an endogenous SNAT1 substrate, and showed that uptake of alanine is essential for T-cell mitogenesis. I also saw Nef- and Vpu-independent regulation of numerous other transmembrane transporters, suggesting a general paradigm for HIV-mediated manipulation of nutrient uptake and cellular metabolism. I now wish to (1) characterise the roles of alanine and SERINC3/5 in T-cell amino acid metabolism; (2) discover new metabolic pathways targeted by HIV in T-cells; and (3) identify key amino acid transporters and metabolic pathways required for T-cell mitogenesis.

Along with alanine transport by SNAT1, ER-resident SERINC proteins incorporate serine into membrane phospholipids. In the 1st part of my project, I will therefore use radiolabelled amino acid transport and stable isotope-labelled metabolite tracing techniques to elucidate the role of alanine in T-cell metabolism, and determine whether cell surface SERINC3/5 act as T-cell amino acid incorporators or transporters. In the 2nd part of my project, I will use Antibody-Free Magnetic Cell Sorting (AFMACS) to isolate HIV-infected T-cells, apply a comprehensive, unbiased metabolomic approach to identify dysregulated metabolic pathways, and determine the mechanisms and viral genes responsible. Finally, in the 3rd part of my project, I will develop a combined plasma membrane proteomic and CRISPR-based genetic screening strategy to determine which transmembrane transporters and metabolic pathways are required for T-cell mitogenesis, focussing on mechanisms for glutamine and net amino acid uptake.

The primary beneficiaries from my results will be other researchers in the fields of retrovirology, immunology, proteomics and metabolism. In addition to specific results, I will generate a significant quantity of data that could be shared for added benefit, and will provide a useful resource for the research community. An example of this will be the cell surface proteomic map of activated primary human CD4+ T-cells.

In addition, I predict that specific materials generated during the project will be of use to the wider community. As an example, I will construct an HIV virus suitable for Antibody-Free Magnetic Cell Sorting (AFMACS), enabling magnetic selection of HIV-infected cells for use in downstream applications. Based on the number of requests I have previously received for AFMACS plasmids, I anticipate that the relevant molecular clones will be in high demand. It is possible (but not anticipated) that, like AFMACS, some new materials will have the potential for commercialisation, to the benefit of Cambridge Enterprise (the commercialisation arm of the University of Cambridge).

Although my proposal involves basic science rather than clinically directed research, it is focused on areas of particular relevance to human disease, with the ultimate aim to deliver novel therapeutic approaches to the clinic. Metabolic pathways targeted by HIV represent candidate targets for antiviral therapy, because some host proteins may be essential for viral replication but not cellular survival. More immediately, cell surface transporters are readily druggable targets, and cell type specific expression offers the opportunity for focussed immunomodulatory therapies targeting mitogenic pathways such as mTOR in T-cells.

In a wider context, my time in Matthew's Vander Heiden's laboratory in MIT will provide a world-class training in techniques and approaches for the study of metabolism. I will use this as a springboard to further develop my research programme when I return to Cambridge. There is a growing interest in metabolism within the Department of Medicine and on the campus, and I anticipate that further collaborative and inter-disciplinary projects will be readily forthcoming, with beneficial cross-fertilisation of the research of other groups. I will seek to disseminate my research skills as well as data and, if this application is successful, the first beneficiary of this will be my RA.