Kinetics of Proteolytic Capsid Disassembly and RNA Secondary Structure Dynamics During Genome Release from a Single-Stranded RNA Virus (2016)

Undergraduate: Amanda Carew

Faculty Advisor: Kevin Weeks
Department: Chemistry

---

Single-stranded RNA viruses are important pathogens that cause hepatitis, HIV and ebola. The RNA genome provides the code for viral proteins and also adopts secondary and tertiary structures that contain additional information necessary for numerous replicative functions. One of the first steps in viral infection is release of the RNA genome from the capsid shell. The RNA genome may play an important role in capsid disassembly as well as undergoing secondary structural changes itself during this process. Satellite tobacco mosaic virus (STMV) is a single-stranded RNA virus with a well characterized genome and protein capsid. The goal of this study was to evaluate RNA secondary structure changes during genome release from STMV during proteolytic viral capsid disassembly. The capsid of STMV was digested by a protease and capsid disassembly was assessed by protein electrophoresis. Near complete capsid disassembly was seen in1 minute. When the RNA genome was evaluated using size-sensitive gel electrophoresis, an RNA conformational change occurred at approximately 30 minutes and was dependent on temperature but independent of protease concentration. These data are consistent with a mechanism in which the capsid is rapidly digested by protease and then there is a first-order (unimolecular) conformational change by the STMV RNA genome. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) was also used to characterize the secondary structural changes of the STMV genome