UL5, A protein required for HSV DNA synthesis

Genetic analysis, overexpression in Escherichia coli, and generation of polyclonal antibodies

Liang Zhu, Sandra K. Weller

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The mutations in two DNA-negative is mutants of herpes simplex virus type 1 (HSV-1), tsK13 and tsM19, have been previously mapped to a 2.0-kb fragment (coordinates 0.095-0.108) at the left end of the genome (S. Weller, D. Aschman, W. Sacks, D. Coen, and P. Schaffer, 1983, Virology 130, 290-305). Sequence analysis of the HSV-1 genome has revealed the existence of two open reading frames, UL5 and UL6, within this fragment (D. McGeoch, M. Dalrymple, A. Dolan, D. McNab, L. Perry, P. Taylor, and M. Challberg,1988, J. Virol. 62, 444-453). In this paper we report fine mapping and sequence analysis of the mutations in tsK13 and tsM19 which unambiguously localize the mutations to UL5, predicted to encode a 99-kDa polypeptide. The mutation in tsK13 was shown to result in a single amino acid substitution, Pro236 to Leu, whereas tsM19 contains two substitutions, Pro236 to Ser and Ala249 to Val. Thus, both mutants are altered in Pro236. Temperature-shift experiments indicated that the UL5 gene product is required continuously during viral DNA synthesis, suggesting a direct role for the 99K protein in viral DNA synthesis. The UL5 gene product was overexpressed in Escherichia coli and used to generate polyclonal antibodies which detected proteins in HSV-1-infected cell extracts from 4 hr postinfection. Although a faint band of the predicted size (99 kDa) was observed, the majority of the immunoreactive material migrated as smaller bands which represent either proteolytic degradation during extraction or post-translational proteolytic modification of the UL5 gene product. Indirect immunofluorescence staining revealed that the UL5 gene product localizes to the nucleus in two patterns: diffuse staining throughout the nucleus and in discrete globules which appear at the periphery of the nucleus. Sequence analysis of the UL5 gene predicts that DNA 99-kDa protein contains a consensus sequence for an ATP binding site. Poss ible roles of this protein in viral synthesis are discussed.

Original languageEnglish (US)
Pages (from-to)366-378
Number of pages13
JournalVirology
Volume166
Issue number2
DOIs
StatePublished - 1988
Externally publishedYes

Fingerprint

Escherichia coli
Human Herpesvirus 1
Antibodies
DNA
Sequence Analysis
Mutation
Genes
Viral DNA
Proteins
Genome
Staining and Labeling
Virology
Consensus Sequence
Viral Proteins
Amino Acid Substitution
Post Translational Protein Processing
Indirect Fluorescent Antibody Technique
Cell Extracts
Open Reading Frames
Adenosine Triphosphate

ASJC Scopus subject areas

  • Virology

Cite this

UL5, A protein required for HSV DNA synthesis : Genetic analysis, overexpression in Escherichia coli, and generation of polyclonal antibodies. / Zhu, Liang; Weller, Sandra K.

In: Virology, Vol. 166, No. 2, 1988, p. 366-378.

Research output: Contribution to journalArticle

@article{8edf95c732864f7b8db429ba8a206fc1,
title = "UL5, A protein required for HSV DNA synthesis: Genetic analysis, overexpression in Escherichia coli, and generation of polyclonal antibodies",
abstract = "The mutations in two DNA-negative is mutants of herpes simplex virus type 1 (HSV-1), tsK13 and tsM19, have been previously mapped to a 2.0-kb fragment (coordinates 0.095-0.108) at the left end of the genome (S. Weller, D. Aschman, W. Sacks, D. Coen, and P. Schaffer, 1983, Virology 130, 290-305). Sequence analysis of the HSV-1 genome has revealed the existence of two open reading frames, UL5 and UL6, within this fragment (D. McGeoch, M. Dalrymple, A. Dolan, D. McNab, L. Perry, P. Taylor, and M. Challberg,1988, J. Virol. 62, 444-453). In this paper we report fine mapping and sequence analysis of the mutations in tsK13 and tsM19 which unambiguously localize the mutations to UL5, predicted to encode a 99-kDa polypeptide. The mutation in tsK13 was shown to result in a single amino acid substitution, Pro236 to Leu, whereas tsM19 contains two substitutions, Pro236 to Ser and Ala249 to Val. Thus, both mutants are altered in Pro236. Temperature-shift experiments indicated that the UL5 gene product is required continuously during viral DNA synthesis, suggesting a direct role for the 99K protein in viral DNA synthesis. The UL5 gene product was overexpressed in Escherichia coli and used to generate polyclonal antibodies which detected proteins in HSV-1-infected cell extracts from 4 hr postinfection. Although a faint band of the predicted size (99 kDa) was observed, the majority of the immunoreactive material migrated as smaller bands which represent either proteolytic degradation during extraction or post-translational proteolytic modification of the UL5 gene product. Indirect immunofluorescence staining revealed that the UL5 gene product localizes to the nucleus in two patterns: diffuse staining throughout the nucleus and in discrete globules which appear at the periphery of the nucleus. Sequence analysis of the UL5 gene predicts that DNA 99-kDa protein contains a consensus sequence for an ATP binding site. Poss ible roles of this protein in viral synthesis are discussed.",
author = "Liang Zhu and Weller, {Sandra K.}",
year = "1988",
doi = "10.1016/0042-6822(88)90507-7",
language = "English (US)",
volume = "166",
pages = "366--378",
journal = "Virology",
issn = "0042-6822",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - UL5, A protein required for HSV DNA synthesis

T2 - Genetic analysis, overexpression in Escherichia coli, and generation of polyclonal antibodies

AU - Zhu, Liang

AU - Weller, Sandra K.

PY - 1988

Y1 - 1988

N2 - The mutations in two DNA-negative is mutants of herpes simplex virus type 1 (HSV-1), tsK13 and tsM19, have been previously mapped to a 2.0-kb fragment (coordinates 0.095-0.108) at the left end of the genome (S. Weller, D. Aschman, W. Sacks, D. Coen, and P. Schaffer, 1983, Virology 130, 290-305). Sequence analysis of the HSV-1 genome has revealed the existence of two open reading frames, UL5 and UL6, within this fragment (D. McGeoch, M. Dalrymple, A. Dolan, D. McNab, L. Perry, P. Taylor, and M. Challberg,1988, J. Virol. 62, 444-453). In this paper we report fine mapping and sequence analysis of the mutations in tsK13 and tsM19 which unambiguously localize the mutations to UL5, predicted to encode a 99-kDa polypeptide. The mutation in tsK13 was shown to result in a single amino acid substitution, Pro236 to Leu, whereas tsM19 contains two substitutions, Pro236 to Ser and Ala249 to Val. Thus, both mutants are altered in Pro236. Temperature-shift experiments indicated that the UL5 gene product is required continuously during viral DNA synthesis, suggesting a direct role for the 99K protein in viral DNA synthesis. The UL5 gene product was overexpressed in Escherichia coli and used to generate polyclonal antibodies which detected proteins in HSV-1-infected cell extracts from 4 hr postinfection. Although a faint band of the predicted size (99 kDa) was observed, the majority of the immunoreactive material migrated as smaller bands which represent either proteolytic degradation during extraction or post-translational proteolytic modification of the UL5 gene product. Indirect immunofluorescence staining revealed that the UL5 gene product localizes to the nucleus in two patterns: diffuse staining throughout the nucleus and in discrete globules which appear at the periphery of the nucleus. Sequence analysis of the UL5 gene predicts that DNA 99-kDa protein contains a consensus sequence for an ATP binding site. Poss ible roles of this protein in viral synthesis are discussed.

AB - The mutations in two DNA-negative is mutants of herpes simplex virus type 1 (HSV-1), tsK13 and tsM19, have been previously mapped to a 2.0-kb fragment (coordinates 0.095-0.108) at the left end of the genome (S. Weller, D. Aschman, W. Sacks, D. Coen, and P. Schaffer, 1983, Virology 130, 290-305). Sequence analysis of the HSV-1 genome has revealed the existence of two open reading frames, UL5 and UL6, within this fragment (D. McGeoch, M. Dalrymple, A. Dolan, D. McNab, L. Perry, P. Taylor, and M. Challberg,1988, J. Virol. 62, 444-453). In this paper we report fine mapping and sequence analysis of the mutations in tsK13 and tsM19 which unambiguously localize the mutations to UL5, predicted to encode a 99-kDa polypeptide. The mutation in tsK13 was shown to result in a single amino acid substitution, Pro236 to Leu, whereas tsM19 contains two substitutions, Pro236 to Ser and Ala249 to Val. Thus, both mutants are altered in Pro236. Temperature-shift experiments indicated that the UL5 gene product is required continuously during viral DNA synthesis, suggesting a direct role for the 99K protein in viral DNA synthesis. The UL5 gene product was overexpressed in Escherichia coli and used to generate polyclonal antibodies which detected proteins in HSV-1-infected cell extracts from 4 hr postinfection. Although a faint band of the predicted size (99 kDa) was observed, the majority of the immunoreactive material migrated as smaller bands which represent either proteolytic degradation during extraction or post-translational proteolytic modification of the UL5 gene product. Indirect immunofluorescence staining revealed that the UL5 gene product localizes to the nucleus in two patterns: diffuse staining throughout the nucleus and in discrete globules which appear at the periphery of the nucleus. Sequence analysis of the UL5 gene predicts that DNA 99-kDa protein contains a consensus sequence for an ATP binding site. Poss ible roles of this protein in viral synthesis are discussed.

UR - http://www.scopus.com/inward/record.url?scp=0023689074&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023689074&partnerID=8YFLogxK

U2 - 10.1016/0042-6822(88)90507-7

DO - 10.1016/0042-6822(88)90507-7

M3 - Article

VL - 166

SP - 366

EP - 378

JO - Virology

JF - Virology

SN - 0042-6822

IS - 2

ER -