Presentation description

Sindbis virus is a single-stranded, positive-sense RNA virus composed of two main parts: nonstructural proteins (NSP) and structural proteins (SP). Nonstructural proteins code for the replication machinery while structural proteins code for Capsid proteins and Glycoproteins, which begin the encapsidation process and help form the mature viral particle, respectively. Sindbis virus is commonly used in lab settings due to its ability to be engineered as a transgene delivery vector. This transgenic system is characterized by the separation of NSP and SP, allowing the NSP to be paired with a transgene of interest and the SP to be provided in trans, making Sindbis virus safer to handle. Typically, Capsid proteins bind and package the NSP-transgene RNA into viral particles. That virus is used to infect new cells with the SPs provided in trans, thus providing all components needed to generate new viral particles. Recently, our lab has seen evidence of Capsid proteins binding to and packaging capsid RNA, leading to defective viral particles and a non-functioning system. Research has shown Capsid proteins bind to GC-rich hairpin loops on the target RNA to begin the packaging process. Here, we utilize codon optimization to synthesize capsid RNA sequences with minimal GC content in an attempt to decrease unwanted capsid RNA packaging. Following the packaging of the virus with both wild-type and codon-optimized capsid RNA, we quantified the presence of capsid RNA using RT-qPCR to determine if codon optimization does, in fact, decrease capsid packaging as hypothesized. Our results showed non-detectable differences in capsid quantification between the wild-type and codon-optimized versions, suggesting there are alternative mechanisms leading to capsid RNA packaging. Although our data did not display the desired effect of less unwanted RNA packaging, it narrows down the pool of possibilities to what leads to the unwanted capsid RNA packaging.