Capsid assembly mechanisms of Kaposi Sarcoma associated herpesvirus

About This Grant

Project Summary Capsid assembly is an essential step in production of an infectious herpesvirus, yet little is known on how the process begins and the protein oligomeric states and intermediate interactions required to complete the final mature capsid. Disruptions in capsid assembly result in aberrant capsids unable to assemble or package viral DNA, resulting in noninfectious virus. The major proteins involved in herpesvirus capsid assembly are generally conserved across alpha, beta, and gammaherpesvirus subfamilies which provides an opportunity to develop pan-herpesvirus therapeutics. Yet to date, little has been done to capitalize on this process; furthermore, most capsid assembly research focuses on alphaherpesvirus, HSV-1. This proposal focuses on understanding capsid assembly of gammaherpesvirus, Kaposi sarcoma herpesvirus (KSHV), the leading cause of cancer in HIV positive individuals. I will develop an in-vitro capsid assembly assay to monitor capsid kinetics and oligomeric states required for capsid nucleation and maturation. I hypothesize KSHV capsid assembly is a highly dynamic process requiring multiple conformations of capsid protein intermediates to generate a capsid capable of packaging a large DNA genome. I will purify KSHV capsid proteins via baculovirus expression and systematically determine the proteins required for capsid formation and kinetics via dynamic light scattering and negative stain electron microscopy. In addition, I will evaluate the oligomeric states of capsid proteins individually and protein intermediates to determine 1. The proteins required for capsid nucleation and 2. How KSHV scaffold oligomerizes to produce unique B-capsid morphologies. I further hypothesize that the divergence of SCP between subfamilies is a result of variations in the capsid assembly pathway. For example, it is thought SCP is not required for alphaherpesvirus capsid assembly but is required for gamma and betaherpesviruses. I hypothesize SCP is required for efficient C-capsid assembly, as loss of SCP results in decreased infectious virus and C-capsid formation in alpha, beta, and gammaherpesvirses; however, the exact mechanism is unclear and seems dependent on cellular environment. I will use the in-vitro capsid assemble assay and live virus mutagenesis to study the function of SCP in capsid maturation. I will utilize previously generated KSHV SCP null viruses to quantify capsid structural integrity with atomic force microscopy to determine at what stage capsid assembly stalls, as well as the role of phosphorylation on SCP’s function. These aims will highlight the similarities and difference between alpha and gammaherpevirus viruses, providing the evidence required for inhibitor development and a scalable in-vitro assay for testing inhibitors. I will develop a robust skillset in biophysical techniques which coupled with my expertise in molecular virology will allow me to successful complete these aims and develop the skills required to run my own independent research lab.