Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry.

ABSTRACT: Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2'-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor- or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were capture

SUBMITTER: Senkevich TG 

PROVIDER: S-EPMC5599757 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature