Identifying regulators of HIV-1 cell-to-cell spread at virological synapses

Human Immunodeficiency Virus Type-1 (HIV-1), the cause of the global AIDS pandemic, replicates primarily in cells of the immune system called CD4+ T cells. An important property of CD4+ T cells is their ability to interact with other immune cells and form close, but transient physical contacts as a means to communicate. During these normal interactions, CD4+ T cells respond to contact from another cell by transmitting signals into their interior to relay information from the exterior, ensuring that they respond appropriately. This is essential for correct functioning of the immune system. Previous research by us and others has shown that HIV-1 can hijack the inherent ability of immune cells to form close contacts in order to move quickly from one cell to another when one of the cells is infected with virus, thereby spreading infection by cell-cell means. Exactly how this is regulated is unknown, but we have recently generated new data that suggests HIV-1 infected cells receive specific signals during contact with uninfected cells, and that this directs the infected cell to respond by rearranging its interior and targeting virus assembly to the contact site. In order to better understand this process we wish to determine exactly what these signals are, how they are initiated and terminated, and how they control HIV-1 replication and spread. In addition, because virus assembly and budding during cell-to-cell spread is an energy-dependent process, it is likely to require cellular powerhouses called mitochondria. Notably, mitochondria are dramatically rearranged in HIV-1 infected cells during cell-cell contact. Therefore, in a related line of research we will also determine what role mitochondria play in HIV-1 transmission. From these studies we aim to gain a greater understanding of how HIV-1 replicates and spreads in HIV-1-infected people, and how normal cellular processes are hijacked by viruses for their own means. The overarching aim of this research is to identify new cellular factors that are required for cell-to-cell transmission of HIV-1 and that may be targeted by drugs in order to limit HIV-1 replication and halt progression to AIDS.

Human Immunodeficiency Virus Type-1 (HIV-1) disseminates rapidly between CD4+ T cells by cell-to-cell transmission at intercellular contacts virological synapses (VS), but how this is regulated is unclear. To address this we propose to couple functional virology and cell biology with proteomics to identify key regulators and signalling pathways implicit in HIV-1 spread. We will use validated cell-to-cell transmission assays to measure HIV-1 transfer between T cells, including quantitative real-time PCR, flow cytometry and reporter assays thereby generating data amenable to robust statistical analysis. HIV-1 infected cells will be treated with chemical inhibitors and putative regulators will be inhibited with RNAi or dominant-negative mutants and we will measure loss of function to identify key proteins and pathways. Signalling pathways will be activated using a reductionist approach of cross-linking specific surface receptors and the effect on T cell polarisation and HIV-1 recruitment will be examined. State-of-the art proteomics analysis will be used to identify novel regulators. We will also perform live-cell imaging of HIV-1 infected cells to follow dynamic processes in real-time. Our working hypothesis is that cell-to-cell transmission of HIV-1 at VS is an energy-dependent, regulated process triggered by cell-cell contact that activates signalling pathways to facilitate efficient viral transmission. The broad aims of this project are to:
1. identify receptors and signalling pathways regulating HIV-1 spread at VS and concomitant T cell polarisation
2. determine how VS are initiated and terminated
3. examine the requirement for T cell polarisation at the VS
4. identify cellular and viral factors implicit in HIV-1 cell-to-cell spread and to investigate strategies of inhibition.
The over-arching aim of this research is to identify novel, potential therapeutic targets to interfere this mode of retroviral dissemination to limit progression to AIDS.

This project aims to increase our understanding of how Human Immunodeficiency Virus type-1 (HIV-1) is transmitted between susceptible CD4+ T cells, and to identify key cellular proteins that regulate and control HIV-1 cell-to-cell spread. The over-arching aim of this work is to identify potentially druggable targets that may be amenable to future therapeutic targeting to limit cell-to-cell spread of HIV-1 in vivo. HIV-1 infection is life-long and cannot be cured. Humoral, cell-mediated and innate immune responses are unable to stop HIV-1 replication in vivo. Antiretroviral therapy (ART) can control viral replication but it is not always effective, and groups of patients have been identified who progress much quicker than others to AIDS even when on ART. Moreover, cell-to-cell spread can persist despite ART. Thus, increasing evidence suggests that cell-to-cell transmission maybe an important contributing factor to HIV-1 pathogenesis, immune evasion and persistence in vivo. From the work proposed in this application we seek to better understand how HIV-1 disseminates by cell-to-cell spread and how this is regulated to identify new, potential drug targets. Our research will have an impact through providing insight into HIV-1 pathogenesis.
The following groups are identified as potential, non-academic beneficiaries of our research in the short and long term:
1. HIV infected patients seeking or on treatment
2. HIV/AIDS education and support groups
3. School children engaged in science education programs and secondary students.
During the 3yr period of this grant we aim to identify key regulators of cell-to-cell spread that we hope to follow up in subsequent studies to determine their susceptibility to therapeutic intervention for which we will seek by future funding. In the longer-term we wish to assess the inhibitory effects of therapeutics on cellular pathways that we identify as important for HIV dissemination. This will be achieved by evaluating compounds that block activation of signalling pathways that we identify as important for HIV transmission during the lifetime of this grant (e.g. using tyrosine kinase inhibitors) and by performing small molecule compound screening to identify novel inhibitors in future work. Although direct clinical benefits to HIV infected individuals are unlikely to be realised over the period of our current grant proposal, we aim to increase our understanding of HIV pathogenesis as an important stepping-stone to realising our broad aims in the longer term.
Our research activities will also be of benefit to HIV/AIDS education/support groups and school children engaged in science education programs. These are short-term aims that can be realised over the course of the project. For example, we will pursue educational workshops and present talks to HIV/AIDS support groups (e.g. Terrance Higgins Trust); participate in science outreach programs at primary schools to engage students in science via hands-on related activities; and host secondary school work experience students who benefit from seeing real laboratory science in action. In these ways we aim to enthuse the next generation of scientists about virology and careers in biomedical science, whilst educating about HIV/AIDS and discussing some of research at a level appropriate to their scientific knowledge.