scholarly journals Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription.

1987 ◽  
Vol 7 (1) ◽  
pp. 7-14 ◽  
Author(s):  
S S Broyles ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Transcription Initiation ◽  
Deae Cellulose ◽  
Polymerase Activity ◽  
Nucleoside Triphosphate ◽  
Early Genes ◽  
Dependent Atpase ◽  
Virus Rna ◽  
High Concentrations

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.

Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription

1987 ◽  
Vol 7 (1) ◽  
pp. 7-14
Author(s):  
S S Broyles ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Transcription Initiation ◽  
Deae Cellulose ◽  
Polymerase Activity ◽  
Nucleoside Triphosphate ◽  
Early Genes ◽  
Dependent Atpase ◽  
Virus Rna ◽  
High Concentrations

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.

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

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Tatiana G. Senkevich ◽  
George C. Katsafanas ◽  
Andrea Weisberg ◽  
Lisa R. Olano ◽  
Bernard Moss
Keyword(s):  
Mass Spectrometry ◽  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Transcription Initiation ◽  
Nuclear Dna ◽  
Binding Protein ◽  
Viral Proteins ◽  
Animal Cells ◽  
Host Proteins ◽  
Viral Dna

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.

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