Extrinsic and Intrinsic Properties of ssDNA Packaging in Mircrovirdae

Abstract

Viral systems across genera have evolved diverse mechanisms that ensure successful encapsidation and organization within virions. Genome packaging requires coordination between trans-acting elements that transport and package the genome; and cis-acting genetic elements that aid in genetic regulation but may also assist in folding and organization. My dissertation focuses on elements that regulate packaging within the øX174-like, G4-like and ⍺3-like microvirus clades.Chapter 1 is a literature review of diverse packaging mechanisms amongst bacteriophage and eukaryotic viruses. Chapter 2 focuses on the genome organization function of the ssDNA binding protein J, that guides and organizes the genome within capsids. Analysis of mutants that have interchanged J genes between clades, display altered genome organization that leads to various packaging defects. Results demonstrate that mutants produced fully packaged uninfectious particles unable to attach to host-cells. Thus, internal organization of the genome can affect properties of the surface of the virion necessary for attachment and penetration. Chapter 3 focuses on the genetic diversification of ⍺3-like microviruses that impacts packaging, such as genome length and temperature sensitivity. Although, capsid volumes are comparable in size between øX174-like, G4-like and ⍺3-like viruses, ⍺3 has the largest genome and is the most cold-sensitive. Due to temperature influencing DNA persistence length, it was hypothesized that ⍺3 is more prone to packaging errors at low temperature. Mutations that expanded the temperature growth limit were isolated and characterized. The results are consistent with this hypothesis, demonstrating that low-temperature adaptation can lead to species diversification. Chapter 4 focuses on periodic sequences in the øX174 genome hypothesized to regulate ssDNA organization within virions. The hypothesis was tested by tracking DNA replication and packaging in a co-infection, between a mutant with modified periodic sequences and wild-type øX174. Results showed that DNA replication was comparable to wild-type. However, at low temperature the mutant produced significantly less progeny virions. This indicates the genome has innate properties that regulate packaging. Together, this work furthers our understanding of ssDNA microvirus packaging regulation. The work reported herein, provides insight into the impact of genome organization on the biophysical properties of the microvirus virion. As well as, insights into the varying role of protein A* protein between microvirus clades; and lastly, ssDNA packaging is under cis-acting regulation which was previously unknown and may be reflected in other ssDNA virus systems.