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 captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate- and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases IIα and IIβ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected.IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNA-dependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases IIα and IIβ and PCNA, which were found associated with nascent DNA.

Keywords: DNA binding proteins; DNA replication; iPOND; poxvirus; transcription factors; vaccinia virus.

FIG 1

Visualization of newly labeled nuclear and cytoplasmic DNA by confocal microscopy. (A) Approximately 80% confluent A549 cells were infected with VACV at a multiplicity of infection of 10 PFU per cell. Uninfected (un) cells and infected cells at the indicated times were incubated for 1 h with 10 μM EdU and then fixed, permeabilized, and reacted with Alexa Fluor 488 azide. The I3 single-strand binding protein was visualized by staining with a MAb, followed by an anti-mouse secondary antibody conjugated to Alexa Fluor 594. DAPI was used to stain total DNA. The cells were viewed with a confocal microscope, and maximum projections are shown. Each subpanel is divided into quadrants: upper left, DAPI (blue); upper right, EdU (green); lower left, I3 (red); and lower right, merge. (B) Cells were infected for 3.5 h and incubated for 10, 20, or 30 min with EdU as for panel A.

FIG 2

Localization of nascent viral DNA relative to the ER. A549 cells were infected with VACV at a multiplicity of infection of 10 PFU/cell. At 3.5 h after infection, the cells were incubated for 10 min with EdU and then fixed, permeabilized, and reacted with Alexa Fluor 488 azide. The cells were then incubated with rabbit polyclonal antibody to calnexin (Santa Cruz), followed by secondary antibody conjugated to Alexa Fluor 594 and DAPI. The image was made with Huygens deconvolution software and analyzed with Imaris 8.4.1.

FIG 3

Superresolution images of nascent DNA. A549 cells infected with VACV for 3.5 h were incubated for 10 (A) or 60 (B) min with EdU, fixed, permeabilized, and reacted with Alexa Fluor 488 azide. In addition, the cells were stained with a MAb to I3, followed by a secondary antibody conjugated to Alexa Fluor 594. The images were made using Huygens deconvolution software and analyzed with Imaris 8.4.1. Scale bars are shown. A higher-magnification image is shown in the inset.

FIG 4

Transmission electron microscopy of EdU-labeled cells. (A) A549 cells were infected with VACV and at 3.5 h were incubated with EdU for 30 min. The cells were fixed and permeabilized with 0.1% Triton X-100 and reacted with biotin-azide, followed by streptavidin–6-nm gold. (B) Same as panel A, except that biotin-azide was omitted as a control. (C) A549 cells were infected and labeled with EdU as for panel A. The cells were then permeabilized with 0.05% saponin and reacted with biotin-azide. The cells were cryosectioned and incubated with streptavidin–10-nm gold. (D) Same as panel C, except that biotin-azide was omitted as a control. Nu, nucleus; Go, Golgi apparatus; Mi, mitochondria.

FIG 5

Kinetics of VACV DNA synthesis in the absence (−) and presence (+) of EdU. A549 cells were infected with VACV for 6 h in duplicate. At 3.5 h, EdU (10 μM) was added to one portion of the cells. At the time points shown, the cells were harvested, and the amounts of VACV DNA and host actin were determined by ddPCR.

FIG 6

iPOND protocol. Confluent A549 cells were infected with VACV, incubated with EdU, and reacted with biotin-azide, and DNA-protein complexes were isolated as depicted. TX-100, Triton X-100; RT, room temperature.

FIG 7

Detection of I3 by Western blotting of proteins associated with affinity-purified EdU-biotin complexes. (A) Gel electrophoresis of deproteinized DNA from the 1% SDS-insoluble and -soluble fractions as shown in Fig. 6. (B) Proteins eluted from streptavidin beads were resolved by SDS-PAGE, electroblotted onto a nitrocellulose membrane, probed with a MAb to the VACV I3 protein, and detected by chemiluminescence. The control (−click) and experimental (+click) samples are indicated. The masses of marker proteins are shown on the right, with the position of monomeric I3 indicated by the arrow. The band of about 62 kDa is presumed to be an I3 dimer.

FIG 8

Relative abundances of viral and host proteins. The average XIC peptide areas for each protein were determined from experiment 1 in Tables 2 and 3. Blue, viral DNA replication proteins; yellow, viral transcription proteins; green, host proteins. The diameters of the circles are proportional to the number of experiments with wild-type virus in which the proteins met the inclusion criteria.