Virus cell biology
We aim to understand aspects of the cellular and molecular mechanisms that underlie the replication of mammalian viruses, specifically how viruses enter target cells, and how new virus particles are assembled and released from infected cells. We currently use HIV-1 and the related simian viruses as models where detailed understanding of cell biology may have application in the development of novel inhibitors or treatments.
Virus entry and productive infection generally require the expression of specific cell surface receptor molecules on target cells. In many cases, virus-receptor binding is followed by endocytosis and viral penetration from within intracellular organelles, usually endosomes. For some viruses, including HIV, the reactions that drive membrane fusion are initiated when the virus engages cell surface receptors – for HIV the co-receptors CD4 plus a chemokine receptor (CCR5 or CXCR4). These infection events can be abrogated by down regulating the cell surface expression of the receptor either by specifically inhibiting synthesis or inducing receptor internalisation and removal from the cell surface. For example, small molecules that inhibit the synthesis of specific cell surface receptors can, in culture systems at least, prevent HIV infection. Agonistic receptor-binding ligands can also be used to induce receptor internalisation and inhibit infection. We are trying to understand the cellular and molecular mechanisms underlying these processes, in particular the events regulating the cell surface expression and endocytosis of HIV-binding co-receptors CCR5 and CXCR4. As these molecules are G protein coupled receptors, our work may have relevance to understanding the trafficking events controlling the functions of other GPCRs. Moreover, our approaches can be applied to further understand other complex virus receptor systems.
We are using a range of morphological approaches, including live cell imaging and EM, to understand how CCR5 and CXCR4 are recruited to endocytic clathrin coated pits following agonist binding. We are particularly intrigued to understand the role of large clathrin lattice domains in these events. The clathrin domains we have analysed are found on the dorsal surface of cells and hence do not appear to have a role in cell-substrate adhesion (though large lattice structures are also seen on the adherent, ventral surfaces of cells), but their function remains to be determined. We also aim to determine how CCR5 molecules in particular are rapidly sorted in early endosomes for delivery to recycling endosomes, and the possible role of co-internalised beta-arrestin molecules in these events. Image-based approaches using the new high throughput screening facilities in the LMCB are being developed to identify key components and small molecular antagonists.
HIV and SIV assembly requires the key viral structural proteins, Gag, Gag-Pol and Env and the viral RNA genome to be bought together at the same place and time within an infected cell to promote the formation of new virus particles. These events occur through interaction with a series of host cell protein machineries. Although a number of the cellular components involved are known, an integrated model of how and when exactly these interactions occur is not yet available. We have previously defined signals in the HIV and SIV envelope glycoproteins (Env) that control the distribution of these key viral proteins in infected cells. In part at least, these signals interact with the clathrin-based sorting machinery, but the full repertoire of interactions remains to be established. Importantly, in a simian model, one of these signals is required for viral pathogenesis. Gag sorting to plasma membrane domains also involves the activities of a repertoire of cellular machineries, including several protein complexes involved in membrane protein sorting and trafficking. How exactly these host cell proteins are coordinated to produce new virus particles, and how Env is recruited during the budding process, remains to be established. In differentiated primary macrophages, the viral structural proteins are sorted to a unique intracellularly sequestered plasma membrane domain. This domain, which is continuous with the plasma membrane but is located within the cell, is present in uninfected macrophages, where in some cases at least it contains MHC class II antigens. In HIV infected macrophages, this membrane domain is the principal site for virus assembly, and newly assembled virions are retained within the compartment prior to virus release. These compartments may play a role in the formation and/or function of immune synapses, and can be exploited by the virus for efficient transfer to CD4+ve T cells via so-called virological or infectious synapses. Although EM analysis indicates that new virions are formed at these specialized plasma membrane domains, the features of the membrane that mediate the recruitment of HIV proteins, but are distinct from the cell surface plasma membrane, remain unclear. High through put approaches are being developed to identify key components involved in the formation of these compartments and their ability to facilitate HIV assembly.
HIV particle budding from an infected HeLa cell. Small gold particles label the Gag structural protein p24/p55; the large gold particles label Env. Scale bar = 100 nm. A. Pelchen-Matthews