Live-Cell Imaging of Vaccinia Virus Recombination

PLoS Pathogens

Patrick Paszkowski¹  David H. Evans¹  Ryan S. Noyce¹ 

[1] Department of Medical Microbiology & Immunology and Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada

关键词: DNA recombination;    Recombinant proteins;    DNA-binding proteins;    DNA viruses;    Viral replication;    DNA replication;    Southern blot;    Viral gene expression;   

DOI  :  10.1371/journal.ppat.1005824

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY