Vaccinia Virus and Poxvirology

Amod P. Kulkarni, Lauriston A. Kellaway, Girish J. Kotwal

Poxviruses are one of the most complex of animal viruses and encode for over 150 proteins. The interactions of many of the poxviral-encoded proteins with host proteins, as well as with other proteins, such as transcription complexes, have been well characterized at the qualitative level. Some have also been characterized quantitatively by two hybrid systems and surface plasmon resonance approaches. Presented here is an alternative approach that can enable the understanding of complex interactions with multiple ligands. The example given is that of vaccinia virus complement control protein (VCP). The complement system forms the first line of defense against microorganisms and a failure to appropriately regulate it is implicated in many inflammatory disorders, such as traumatic brain injury, Alzheimer's disease (AD), and rheumatoid arthritis. The complement component C3 is central to the complement activation. Complement regulatory proteins, capable of binding to the central complement component C3, may therefore effectively be employed for the treatment and prevention of these disorders. There are many biochemical and/or immunoassays available to study the interaction of proteins with complement components. However, protocols for many of them are time consuming, and not all assays are useful for multiple screening. In addition, most of these assays may not give information regarding the nature of binding, the number of molecules interacting with the complement component C3, as well as kinetics of binding. Some of the assays may require labeling which may induce changes in protein confirmation. We report a protocol for an assay based on quartz crystal microbalance with dissipation monitoring (QCM-D) technology, which can effectively be employed to study poxviral proteins for their ability to interact with their ligand. A protocol was developed in our laboratories to study the interaction of VCP with the complement component C3 using Q-sense (D-300), equipment based on QCM-D technology. The protocol can also be used as a prototype for studying both proteins and small-sized compounds (for use as anti-poxvirals) for their ability to interact with and/or inhibit the activity of their ligands.