Research Article: Live-Cell Imaging of Vaccinia Virus Recombination

Date Published: August 15, 2016

Publisher: Public Library of Science

Author(s): Patrick Paszkowski, Ryan S. Noyce, David H. Evans, Nels C. Elde.


Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren’t detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories.

Partial Text

Genetic recombination serves an essential role as a mechanism for repairing DNA damage, especially the double-stranded breaks that are produced when the replication machinery encounters single-stranded nicks in template DNA. In the field of virology, recombination was first used to define and map bacteriophage genes [1, 2] and is widely used as a tool for genetically engineering a great diversity of viruses. Recombination also affects poxviruses, as was shown by early work with cowpox, variola and vaccinia viruses (VACV) [3, 4]. It was subsequently used to map VACV genes using both classical and marker rescue methods [5–9] and methods developed in the 1980s [10, 11] are also still widely used to produce genetically modified poxviruses.

These studies provide insights into when recombinant genes can be formed during VACV replication and how that process is affected by the arrangement of the recombining fragments in cis (i.e. on the same genome), or in trans (on different genomes). The technology is somewhat constrained by the limits imposed by the kinetics of virus promoter activation, nevertheless some important general features of poxvirus recombination are illustrated by these studies.




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