N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein

Ruyechan, William T.

doi:10.1128/jvi.62.3.810-817.1988

Cited by 18 publications

(9 citation statements)

References 36 publications

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“…This is the first report of the Chromatography of HSV DNA polymerase, ICP8, and UL37 proteins on SS DNA-agarose. HSV-1-infected cell proteins were isolated by the procedure of Ruyechan (33). Polymerase assays and immunoblots were performed as described in Materials and Methods.…”

Section: Discussionmentioning

confidence: 99%

“…For studies involving the DNA synthesis inhibitor PAA, cells were infected and maintained in the presence of 300 ,ug of PAA per ml. For SS DNA-binding studies, protein extracts were prepared essentially as previously described (33). Briefly, CV-1 cells were infected at a multiplicity of infection of 10, and at 24 hours postinfection (HPI), the infected cells were scraped, pelleted, and quick frozen at -70°C.…”

Section: Construction Of Recombinant Plasmids Plasmid Prb210mentioning

confidence: 99%

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Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame

Shelton

Pensiero

Jenkins

1990

J Virol

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The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between map units 0.527 and 0.552. We have identified and characterized the UL37 protein product in HSV-1-infected cells. The presence of the UL37 protein was detected by using a polyclonal rabbit antiserum directed against an in vitro-translated product derived from an in vitro-transcribed UL37 mRNA. The UL37 open reading frame encodes for a protein with an apparent molecular mass of 120 kDa in HSV-i-infected cells; the protein's mass was assigned on the basis of its migration in sodium dodecyl sulfate-polyacrylamide gels. The UL37 protein is not present at detectable levels in purified HSV-1 virions, suggesting that it is not a structural protein. Analysis of time course experiments and experiments using DNA synthesis inhibitors demonstrated that the UL37 protein is expressed prior to the onset of viral DNA synthesis, reaching maximum levels late in infection, classifying it as a 'yl gene. Elution of HSV-1-infected cell proteins from single-stranded DNA agarose columns by using a linear KCI gradient demonstrated that the UL37 protein elutes from this matrix at a salt concentration similar to that observed for ICP8, the major HSV-1 DNA-binding protein. In addition, computer-assisted analysis revealed a potential ATP-binding domain in the predicted UL37 amino acid sequence. On the basis of the kinetics of appearance and DNA-binding properties, we hypothesize that UL37 represents a newly recognized HSV-1 DNA-binding protein that may be involved in late events in viral replication. * Corresponding author. in cells infected by HSV-1 in the presence of DNA synthesis inhibitors and therefore was initially identified as an early (,) transcript on the basis of the nomenclature at the time for the different temporal classes of HSV genes. In this study, we report on the identification and characterization of the UL37 protein from HSV-1-infected cells. Our results demonstrate that the UL37 ORF encodes a protein with an apparent molecular mass of 120 kDa, which is nonstructural, belongs to the-yl class of HSV genes, and binds to single-stranded (SS) DNA-agarose columns. A search of the predicted UL37 amino acid sequence reveals a potential ATP-binding site, and computer-assisted analysis comparing the UL37 sequence with the entire varicella-zoster virus (VZV) genome demonstrates that the VZV homolog, gene 21, shares 47% similarity at the amino acid level with UL37. While we currently have not identified a specific function for the UL37 protein, we hypothesize, on the basis of the results presented in this report, that the UL37 protein is a newly recognized HSV-1 DNA-binding protein which may play a role either in viral gene regulation or in the processing of newly synthesized viral DNA. MATERIALS AND METHODS Cells and viruses. Vero and CV-1 cells (American Type Culture Collection) and human thymidine kinase-negative (TK-) 143 cells were grown in Eagle's minimal essential medium supplemented with 10% (vol/vol) Serum-Plus (Hazleton Co...

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Section: Discussionmentioning

confidence: 99%

Section: Construction Of Recombinant Plasmids Plasmid Prb210mentioning

confidence: 99%

Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame

Shelton

Pensiero

Jenkins

1990

J Virol

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“…Data from other systems indicate that redox regulation can play a role in the function of DNA-binding factors. This has been observed with transcriptional factors (reviewed in (Bauer et al, 1999;Kim et al, 2002)) and some other SSB proteins including the eukaryotic protein RP-A (You et al, 2000), herpes simplex virus -1 (HSV-1) protein ICP8 ( Knipe et al, 1982;Ruyechan, 1988;Dudas and Ruyechan, 1998), and baculovirus LEF-3 (Mikhailov et al, 2005). Similar to ICP8 and LEF-3, the DBP activities were also inhibited by alkylation with NEM.…”

Section: Discussionmentioning

confidence: 74%

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

2008

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DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His(6)-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.

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“…The ability of ICP8 to interact with nucleic acids has been the subject of extensive study since its initial identification. Work from many laboratories has shown that ICP8 binds preferentially and cooperatively to single-stranded DNA (2,13,42,(45)(46)(47)(48)(49). This interaction is reminiscent of, although not identical to, that observed with the T4 gene 32 protein and the Escherichia coli single-strand-DNA-binding protein, based on results of agarose gel electrophoresis and electron microscopy.…”

mentioning

confidence: 87%

Identification of a Region of the Herpes Simplex Virus Single-Stranded DNA-Binding Protein Involved in Cooperative Binding

Dudas

Ruyechan

1998

J Virol

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We have identified a region of the herpes simplex virus major DNA-binding protein (ICP8) which is involved in cooperative binding to single-stranded DNA. This has been accomplished by analysis of ICP8 which was covalently modified by reaction with the extrinsic fluorophore fluorescein-5-maleimide (FM). Reaction conditions which result in the incorporation of 1 mol of FM per mol of ICP8 have been established. The binding properties of the modified protein were analyzed by polyacrylamide gel shift analysis with model oligonucleotides. This analysis indicates that while intrinsic binding is similar to that observed with unmodified protein, the cooperative binding of the modified protein to single-stranded DNA is significantly altered. Helix-destabilizing assays, whose results are a reflection of cooperative binding, also indicate that this property of ICP8 is decreased upon modification with FM. Mapping of the site of modification by cyanogen bromide cleavage and peptide sequencing has shown that the major site of modification is cysteine 254. This position in the primary structure of ICP8 is distinct from the regions previously shown to be involved in the interaction of this protein with single-stranded DNA.

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N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein

Cited by 18 publications

References 36 publications

Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame

Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

Identification of a Region of the Herpes Simplex Virus Single-Stranded DNA-Binding Protein Involved in Cooperative Binding

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