Robert A. Pugh, PhD

Research Title: Alphavirus Replication Complexes

Project Summary: Dr. Robert Pugh’s project focused on expanding previous findings regarding replication and replication complex formation in model systems to human viruses including Sindbis and Chikungunya. Members of the Ahlquist laboratory previously used Brome mosaic virus, a plant virus and member of the alpha-like virus superfamily, as a model system for studying the viral and host proteins that are involved in formation of the replication complex and viral replication. During his appointment on this training grant, Dr. Pugh was able to develop a system to study alphavirus replication complex formation in mammalian and human cells. He expressed one of the key viral proteins involved in formation of the replication complex, nsP1 from both Sindbis and Chikungunya. Successful expression of nsP1 led to formation of extensions from the plasma membrane that were observable by immunofluorescence using confocal microscopy and cryoelectron microscopy. This allows additional studies to further explore the nature of these structures and key protein-protein interactions that result in the formation of filament-like protrusions from the plasma membrane that will shed insight into how these viruses form complete replication complexes. Dr. Pugh’s work required construction of expression vectors using standard molecular cloning techniques as well as extensive training in cell and tissue culture techniques using mammalian and human cells. Additionally, viral stocks of Sindbis were harvested and used to infect mammalian cells with replicating virus in an effort to obtain high resolution images of alphavirus replication complexes at the plasma membrane using cryoelectron microscopy. Preliminary studies have successfully identified these replication complexes, and members of the Ahlquist laboratory are continuing to work to advance current understanding of how these replication complexes form and to identify novel structures in these complexes.