Genomic RNA control of HIV viral assembly and export

HIV remains a major cause of illness and death worldwide despite major advances in the diversity and efficaciousness of available drugs to treat the virus. HIV is an RNA virus which encapsidates two copies of its RNA genome into each virus particle. This is a process which the virus achieves by a very specific interaction between domains of the major structural viral protein Gag and a region of the viral full length genomic RNA which has folded up to form a uniquely recognisable three dimensional shape. The folding up of the RNA is dependent on the nucleotide sequence of the virus RNA so this particular structure can only be formed by the HIV genome. Many of the details about how the virus assembles itself and captures its genetic material as it leaves the cell are still incompletely understood. Because of this it is a part of the lifecycle which is a currently unexploited therapeutic target. We know many of the cellular and viral proteins involved but how they interact with the RNA genome is still not fully elucidated. We have now found evidence that the virus uses the presence of its genetic material, RNA, as a quality control measure to regulate the efficiency of virus export. Only viruses which contain the RNA genome undergo the correct maturation steps and bud with optimal efficiency from the infected cell. This is important for the virus since a particle that budded out without capturing any genetic material would not be infectious. Our data show that the presence of the RNA controls how the virus assembles and how the virus matures into an infectious particle. The presence of the RNA also seems to ensure correct interactions between viral and cellular proteins which are used to facilitate budding. Much is known about the protein factors involved in virus budding but until now the way the RNA component controls the process has largely been ignored. Our published and preliminary data indicates that there are important interactions between the viral RNA and viral and cellular proteins that can be exploited as new ways to inhibit virus replication. We will analyse this process to establish and characterise specific interactions of the viral RNA with cellular proteins, some of which we have already identified. We will use the most modern techniques for identifying RNA/protein interactions, including some that we have developed ourselves. We will also use super resolution microscopy and crystallography to visualise the core components of a budding complex of the virus and how the different components interact. By understanding these processes fully we will be able to identify new potential targets for drug intervention. We have already exploited part of this process (involving purely viral components) as a drug target and this is the subject of an ongoing collaboration with Glaxo SmithKline. More detailed knowledge of processes involving cellular proteins will provide new targets which the virus will have difficulty escaping from and which will add usefully to the armamentarium of drugs available to treat this infection.

Production of an infectious HIV viral particle requires the inclusion of a dimeric RNA genome. Capture of the genomic RNA (gRNA) by a small number of molecules of the structural protein Gag appears to be the initiating event. However, Gag interacts with only a small proportion of the gRNA and is joined by cellular proteins to form the trafficking ribonucleoprotein complex that arrives at the viral budding site. At the plasma membrane a series of cellular proteins including those of the ESCRT series and associated factors such as ALIX cooperate to facilitate virus export. There is growing evidence that some of these proteins have RNA binding capability which is critically involved in successful virus production and also that the presence of the viral genome facilitates the correct cleavage rates of the protease on the structural proteins. We have identified RNA interactions with some of these factors and there is published evidence that supports others. We plan to take an RNA focussed view of viral production and to seek the interactions of the RNA genome with these factors by structural and functional analyses. We will determine which interactions are essential for virus export and would thus be possible targets for antiviral therapies. Using techniques based on cross linking and immunoprecipitation (CLIP) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) as well as super resolution microscopy and crystallography we aim to build up a picture of the components and exact structure of the encapsidation complex which allows specific packaging of HIV genomic RNA. Our aim is to understand the interactions of the viral genomic RNA with cellular proteins. Ultimately this would lead to development of small molecule screens targeting those interactions analogous to the work we have performed on HIV packaging which has led to a collaboration with GSK seeking inhibitors of the RNA/protein interaction involved in genomic RNA encapsidation.

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 being able to mutate the interacting viral factor or sequence and provide a possible route to highly efficacious treatments with a higher threshold for mutational escape than therapeutic approachees targeting purely virus specific factors and processes. We have a track record of developing new paradigms of antiviral therapy for HIV and 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 health care 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. The current proposal has the potential to create similar advances in technical assays 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.

Whilst not directly contributing to increased public awareness in itself, the appointed researchers and the PIs will take very 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 bioinformatic and IT skills required to perform this work enhances the ability of individuals employed on the project to work, if they wish, in non-academic professions.

Research which 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 RNA protein interactions and structural biological phenomena) and will lead ultimately to quality of life enhancement.

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.