Proteomic Screen for Cellular Targets of the Vaccinia Virus F10 Protein Kinase Reveals that Phosphorylation of mDia Regulates Stress Fiber Formation

Greseth, Matthew D.; Carter, Dominique C.; Terhune, Scott S.; Traktman, Paula

doi:10.1074/mcp.m116.065003

Cited by 9 publications

(7 citation statements)

References 85 publications

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“…Instead, spherical structures of smaller size and greater electron density than immature virions, and lacking any internal structure, were observed (*). Such aberrant virions have been seen before when DNA encapsidation was blocked by the repression of the A32 protein (30) or the impairment of the telomere binding protein I6 (31). These images support the conclusion that, although late proteins are abundant during vΔI3 infections of BSC40 cells, infectious virions are not produced because the viral DNA that does accumulate (5% of WT levels) is subgenomic and cannot be encapsidated.…”

Section: Resultssupporting

confidence: 65%

“…These spherical, electron-dense, aberrant virions lack DNA, have no visible internal structure, and are not infectious. Interestingly, however, BSC40 cells infected with vΔI3 do not contain the crystalloids seen when I6, A32, or the viral A13 protein is defective or absent (30,31,44). These electron-dense crystalloids contain closely packed arrays of viral DNA.…”

Section: Discussionmentioning

confidence: 98%

“…Similarly, no mature virions were seen. However, numerous aberrant virions that closely resembled those seen during tsI6 infections and A32-deficient infections were seen (30,31). I6 is a telomere binding protein that associates with the hairpin telomeres of the monomeric viral genome (41), and A32 is thought to be a pumping ATPase of the FTS-HerA family (42,43).…”

Section: Discussionmentioning

confidence: 99%

“…I6 is a telomere binding protein that associates with the hairpin telomeres of the monomeric viral genome (41), and A32 is thought to be a pumping ATPase of the FTS-HerA family (42,43). When I6 is defective or A32 is absent, viral genomes are not encapsidated (30,31). These spherical, electron-dense, aberrant virions lack DNA, have no visible internal structure, and are not infectious.…”

Section: Discussionmentioning

confidence: 99%

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Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication

Greseth

Czarnecki

Bluma

et al. 2018

J Virol

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Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication. Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.

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Section: Resultssupporting

confidence: 65%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication

Greseth

Czarnecki

Bluma

et al. 2018

J Virol

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“… Save and extract the gz-compressed file into the data folder created in step 1.2.1. NOTE: Alternatively, download example data for human phosphoproteomics searched with MaxQuant from the PRIDE archive (file “Traktman_2013_MaxQuantOutput-full.zip” from https://www.ebi.ac.uk/pride/archive/projects/PXD005246/files 23 ). Save and extract the zip-compressed file in the data folder that was created in step 1.2.1.…”

Section: Protocolmentioning

confidence: 99%

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Schlaffner

Pirklbauer

Bender

et al. 2018

JoVE

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Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

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Multi-omics characterization of the monkeypox virus infection

Huang,

Bergant,

Grass

et al. 2024

Nat Commun

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Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-β pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.

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Proteomic Screen for Cellular Targets of the Vaccinia Virus F10 Protein Kinase Reveals that Phosphorylation of mDia Regulates Stress Fiber Formation

Cited by 9 publications

References 85 publications

Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication

Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Multi-omics characterization of the monkeypox virus infection

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