MRC Transition Support CSF Nicholas Matheson

Approximately 100 years since it was first transmitted to humans, the Human Immunodeficiency Virus (HIV) infects almost 40 million people worldwide, and causes around 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. I previously used proteomics to measure changes in the number and quantity of proteins at the surface of cells infected with HIV, and identified many proteins 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 drugs. I therefore wish to understand why amino acid transporters are targeted by HIV, and the importance of these transporters for "helper 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 helper 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.

During the first 2.5 years of my Clinician Scientist Fellowship, I have: developed a new way of purifying HIV-infected helper T cells; perfected a way to extract amino acids and other "metabolites" from these cells; and developed a transformative approach to measuring transport of amino acids in and out of cells. I therefore now wish to use these techniques to figure out which metabolic processes in cells are manipulated by HIV, and why they are important for the HIV virus and helper T cells.

In particular, they will enable me to: measure amino acid transport by SNAT1 and SERINC3/5 in growing cells; map global changes in metabolites and metabolism in HIV-infected primary CD4+ T cells; and characterise other critical amino acid transporters of helper T cells identified using different, unrelated approaches earlier in my fellowship. Taken together, these data will be a valuable resource for other researchers in the field and will, I hope, ultimately lead to the development of new treatments for patients targeting amino acid transporters in T cells.

Approximately 100 years since its transmission to humans, HIV-1 infects almost 40 million people worldwide, and causes around 1 million AIDS-related deaths every year. I previously used TMT-based plasma membrane proteomics to gain a comprehensive, unbiased overview of cell surface proteins regulated by HIV infection. In addition to known targets, I discovered HIV-mediated downregulation of the amino acid transporter SNAT1, and the putative serine carriers SERINC3/5.

I found SNAT1 to be dramatically upregulated at the surface of activated T cells, identified alanine as an endogenous SNAT1 substrate, and showed that extracellular alanine is essential for T cell mitogenesis. Along with SNAT1 and SERINC3/5, I also observed regulation of other transmembrane transporters, suggesting a general paradigm for HIV-mediated manipulation of nutrient uptake and cellular metabolism.

During the first 2.5 years of my Clinician Scientist Fellowship, I have: developed a reporter virus (HIV-AFMACS) allowing magnetic selection of HIV-infected primary CD4+ T cells; optimised a novel method for metabolite extraction from these cells; and developed a transformative stable isotope-based approach to quantitate amino acid transport.

I therefore now wish to leverage these techniques to further elucidate the metabolic processes manipulated by HIV, and define their importance in HIV pathogenesis and T cell immunobiology. In particular, they will enable me to: quantitate amino acid transport by SNAT1 and SERINC3/5 under normal growth conditions in a comprehensive, unbiased fashion; map global metabolic changes in HIV-infected primary CD4+ T cells; and characterise other critical mitogenic amino acid transporters of CD4+ T cells identified by a combined proteomic-genetic screening approach.

The primary beneficiaries from my results will be other researchers in the fields of retrovirology, immunology and metabolism. I will generate a significant quantity of data that may be shared for added benefit, and will provide a useful resource for the community. To my knowledge, this body of work will represent the first systematic application of stable isotope-based tracing to quantitate amino acid transport. As such, it has the potential to transform the way scientists measure and understand transport of amino acids and other metabolites.

Critically, the novel techniques to quantitate metabolism described in this application are applicable to a wide range of cell biological questions, not just HIV/immunometabolism. I am committed to sharing detailed methods with other researchers, to enhance the efficiency and reproducibility of research in the field. Materials used during this project will also be useful to the wider community, such as the HIV-AFMACS virus. Judging by the number of requests I have previously received for AFMACS reagents, I anticipate that these molecular clones will be in high demand.

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, particularly if they are 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 CD4+ T cells.

In a wider context, my time in Matthew's Vander Heiden's laboratory at MIT has provided me with a world-class training in the study of metabolism. I am now using this as a springboard to further develop my research programme in Cambridge, including contributing to the new Cambridge Institute for Therapeutic Immunology and Infectious Disease (CITIID) Immunometabolic Core facility. 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 therefore disseminate my research skills as well as data, and the first beneficiary of this will be my RA.