Understanding the assembly of simple ssRNA virus nucleocapsids

Lamb, Kristen
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The assembly of simple ssRNA viruses from the initial interactions critical for nucleocapsid formation until the morphogenesis which occurs at each maturation stage of the virus’s “life” can entail a wide range of possibilities at every given stage. By whatever seeding event initiates the nucleocapsid formation, the nucleocapsid, encasing the nucleic acid, then undergoes changes associated with the virus maturation process, which can involve, becoming enveloped, interacting with additional virus/host proteins, becoming subsequently stripped of its envelope, and/or exposure to changes in ion concentration or pH change. My studies focused on two viruses, Hepatitis C Virus (HCV), focusing on the physical characteristics of core protein and in vitro nucleocapsid assembly; and Venezuelan Equine Encephalitis Virus (VEEV), focusing on in vivo formation of pre-viral nucleocapsid structures and briefly the subsequent post-entry nucleocapsid stage. Experimentally, it was found that H124 expressed better in E. coli expression systems than H158 and H36-169, which was attached to MBP. Purified H158 was found to be largely unstructured and exist as a multimer, even at a concentration of 1 mg/mL, making crystallization attempts with it a vain and unprofitable goal. The nucleocapsid-like-particles produced with H158 or H124 both showed size heterogeneity within the reactions, and given the scale of the reactions, projected purification yields would make cryo-EM reconstructions of these impractical. Therefore, focus shifted to VEE nucleocapasids, formed in vivo, enabling the experimental scale and biological relevance to increase substantially. VEE pre-viral nucleocapsid cryo-EM reconstructions were successfully achieved, but at low resolution. Given, again, the heterogeneity of the system at this maturation stage, it is more an insight into one, maybe two, structures,which exist within the population. Thus, more questions are formed for the formation of ssRNA virus nucleocapsid formation.

simple ssRNA virus, cryo-EM, assembly