Investigating barriers to vaccinia virus recombination using combinations of light and electron microscopy

  • Author / Creator
    Kieser, Quinten J
  • While DNA viruses typically replicate in the nucleus of the host cell, poxvirus replication occurs exclusively within cytoplasmic structures known as viral factories. Viral factories are the sites of various processes of the vaccinia virus (VACV) life cycle including replication, morphogenesis and recombination. Recombination is catalyzed by the viral polymerase and, as such, can be detected early in infection alongside DNA replication. However, previous work in our lab showed that recombination between two co-infecting particles is significantly delayed compared to recombination events that occur within a single virus. These delays were attributed to a physical barrier that restricts genetic exchange, a prerequisite for intergenic recombination, until late in infection. These barriers may arise due to multiple characteristics of the viral life cycle. First, it has been shown that each infecting particle gives rise to its own viral factory. Even after the apparent fusion of these individual factories, the genetic content of a single factory remained distinct. Second, each factory is enwrapped with membranes derived from the ER early during infection. It stands to reason that these membranes could persist late into infection and prevent the DNA from two closely apposed factories from mixing. Here I describe the use of light and electron microscopy to investigate the sub-structure of viral factories and the potential constraints they impose on inter-genomic recombination. Initially, we labelled calreticulin, a marker of the endoplasmic reticulum (ER) from which the membranes that enclose viral factories are thought to be derived, and observed staining patterns that suggest that viral factories are surrounded and potentially separated by membrane structures. These observations translated well to initial electron microscopy experiments that showed membrane structures existed, at least to a limited extent, around the periphery of viral factories. Further studies used correlative light and electron microscopy to investigate the membrane ultrastructure associated with recent collision events. Under this system, cell structures, including ER-like membranes and mitochondria, could be observed at the junction of two recently collided factories. However, investigation of the 3D-ultrastructure of a recent collision event showed that these structures existed in only a limited capacity throughout the z-dimension and in a way that would not meaningfully restrict genetic mixing and recombination between closely apposed viruses. Altogether, these studies show that the membrane structures present at the periphery of viral factories early in infection likely play little role in restricting genetic mixing of factories that collide late during infection.

  • Subjects / Keywords
  • Graduation date
    Spring 2020
  • Type of Item
  • Degree
    Master of Science
  • DOI
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