Graham Simmons, PhD
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Over the preceding 30 years many new infectious agents have entered the sphere of human diseases, while others have reemerged, often as drug resistant strains, or have geographically shifted into previously unaffected areas. Many of these agents are enveloped RNA viruses, such as HIV and Hepatitis C, and pose a threat to the blood supply, particularly during the initial transition into the population, before routine testing becomes available.
Enveloped viruses acquire a lipid coat during particle release from infected cells. In order to gain entry into new cells, the virus faces the challenge of fusing this lipid envelope with the membrane of the target cell. Membrane-bound viral fusion proteins studding the outside of the viral particle actuate the process of membrane fusion. By directing viral entry, fusion proteins play an essential role in determining what cell types and tissue become infected. They are also often the primary target for protective antibodies following infection or vaccination.
In order to study the entry process, viral fusion proteins can be incorporated into the lipid coat of modified retroviral particles lacking their own fusion protein. These so-called pseudovirions, mediate attachment and membrane fusion, and hence infection of target cells, in a manner analogous to that observed with authentic virus. Thus, specialized safety containment and practices are no longer required, while the use of reporter genes encoded by the retrovirus genome yields a rapid, and quantitative, readout for infection.
The successful production of pseudovirions for emerging infectious agents allows the creation of assays to further study the entry process, as well as providing a useful tool for identifying and characterizing compounds and antibodies capable of neutralizing viral entry. In collaboration with laboratories experienced at high-throughput screening, pseudovirions, and other approaches, are being utilized by Dr Simmons’ group in order to screen compound libraries for inhibitors of viral entry directed against emerging infectious agents. In addition, pseudovirions are providing the basis for assays to rapidly quantify neutralizing antibody induction in response following actute infection.
Fusion proteins operate by the induction of a cascade of conformational rearrangements within the protein leading up to the mediation of membrane fusion. Understanding the intermediates and steps involved in this ordered sequence of conformational change will open up new areas of development for anti-viral drugs and targeted vaccine studies. Dr Simmons has demonstrated that subsequent to initial receptor interactions, a proteolytic event is required in order for efficient coronavirus and filovirus entry to occur. This cleavage, presumably of the viral fusion protein following receptor-induced conformational change, can either be mediated by exogenous proteases such as trypsin at the cell surface, or within endosomes following viral uptake, by the pH-dependent cellular protease cathepsin L.
Simmons G, Reeves JR, Rennekamp AJ, Amberg SM, Piefer AJ, Bates P. Characterization of severe acute respiratory syndrome-associated coronavirus spike protein mediated viral entry. PNAS 101:4240-4245. 2004.
Temperton NJ, Chan PK, Simmons G, Zambon MC, Tedder RS, Takeuchi Y, Weiss RA. Longitudinally profiling neutralizing antibody response to SARS Coronavirus with pseudotypes. Emerg Infect Dis. 11:411-416. 2005.
Simmons G, Gosalia DN, Rennekamp AJ, Reeves JR, Diamond SL, Bates P. Inhibitors of cathepsin L prevent SARS coronavirus entry. PNAS. 102:11876-11881. 2005.
Amberg SM, Netter RC, Simmons G, Bates P. Expanded tropism and altered activation of a retroviral glycoprotein resistant to an entry inhibitor peptide. J Virol. 80:353-359. 2006.
Simmons G, Rennekamp AJ, Bates P. Proteolysis of SARS-associated coronavirus spike glycoprotein. Adv Exp Med Biol. 581:235-240. 2006.
Kaletsky RL, Simmons G, Bates P. Proteolysis of the Ebola glycoproteins enhances virus binding and infectivity. J Virol. (In Press). 2007.