Evidence for a direct role for both the 175,000- and 110,000-molecular-weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters

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Abstract

We reconstructed the regulated induction of delayed-early (DE) transcription that occurs during herpes simplex virus (HSV) infection by using a transient expression system in which recombinant target genes were cotransfected into Vero cells together with intact activating genes. Plasmids containing clones HSV-1 or HSV-2 immediate-early (IE) genes stimulated by up to 100-fold the expression from recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the DE promoter/regulatory region from the genes for an HSV-2 38,000-molecular-weight (38K) protein and the HSV-1 thymidine kinase. This activation was specific to hybrid genes containing DE regulatory regions since no significant increases in expression were observed in cotransfection experiments with the CAT gene without any promoter region or under the control of a number of other regulatory regions, including an HSV-1 1E regulatory region, the complete or enhancerless early regulatory region of simian virus 40, and an inducible cellular promoter/regulatory region. By using a variety of cotransfected plasmids containing individual or different combinations of HSV-1 or HSV-2 IE genes, we show that of the five known IE genes, two, those coding for the 175K and 110K polypeptides, each possessed the ability to stimulate expression from both DE promoters. Cleavage of the input plasmids within the known coding regions for the 175K and 110K proteins abolished stimulation of DE/CAT gene expression, whereas cleavage outside the coding regions had no effect on stimulation. We conclude that stimulation of CAT expression occurred exclusively by a transactivation mechanism in which the products encoded by these IE genes acted on the DE hybrid constructs at the transcription level. No transcriptional stimulatory function was demonstrated for the IE 68K and 63K proteins, although our results indicate that the IE 12K protein may augment the DE stimulatory activity of the 175K and 110K proteins.

Original languageEnglish (US)
Pages (from-to)751-760
Number of pages10
JournalJournal of Virology
Volume53
Issue number3
StatePublished - 1985

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Immediate-Early Proteins
herpes simplex
Nucleic Acid Regulatory Sequences
transcriptional activation
Simplexvirus
Transcriptional Activation
Chloramphenicol O-Acetyltransferase
Immediate-Early Genes
Molecular Weight
promoter regions
Human Herpesvirus 1
molecular weight
viruses
Human Herpesvirus 2
chloramphenicol acetyltransferase
Human herpesvirus 1
Genetic Promoter Regions
Human herpesvirus 2
Plasmids
genes

ASJC Scopus subject areas

  • Immunology

Cite this

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title = "Evidence for a direct role for both the 175,000- and 110,000-molecular-weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters",
abstract = "We reconstructed the regulated induction of delayed-early (DE) transcription that occurs during herpes simplex virus (HSV) infection by using a transient expression system in which recombinant target genes were cotransfected into Vero cells together with intact activating genes. Plasmids containing clones HSV-1 or HSV-2 immediate-early (IE) genes stimulated by up to 100-fold the expression from recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the DE promoter/regulatory region from the genes for an HSV-2 38,000-molecular-weight (38K) protein and the HSV-1 thymidine kinase. This activation was specific to hybrid genes containing DE regulatory regions since no significant increases in expression were observed in cotransfection experiments with the CAT gene without any promoter region or under the control of a number of other regulatory regions, including an HSV-1 1E regulatory region, the complete or enhancerless early regulatory region of simian virus 40, and an inducible cellular promoter/regulatory region. By using a variety of cotransfected plasmids containing individual or different combinations of HSV-1 or HSV-2 IE genes, we show that of the five known IE genes, two, those coding for the 175K and 110K polypeptides, each possessed the ability to stimulate expression from both DE promoters. Cleavage of the input plasmids within the known coding regions for the 175K and 110K proteins abolished stimulation of DE/CAT gene expression, whereas cleavage outside the coding regions had no effect on stimulation. We conclude that stimulation of CAT expression occurred exclusively by a transactivation mechanism in which the products encoded by these IE genes acted on the DE hybrid constructs at the transcription level. No transcriptional stimulatory function was demonstrated for the IE 68K and 63K proteins, although our results indicate that the IE 12K protein may augment the DE stimulatory activity of the 175K and 110K proteins.",
author = "P. O'Hare and Hayward, {Gary Selwyn}",
year = "1985",
language = "English (US)",
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T1 - Evidence for a direct role for both the 175,000- and 110,000-molecular-weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters

AU - O'Hare, P.

AU - Hayward, Gary Selwyn

PY - 1985

Y1 - 1985

N2 - We reconstructed the regulated induction of delayed-early (DE) transcription that occurs during herpes simplex virus (HSV) infection by using a transient expression system in which recombinant target genes were cotransfected into Vero cells together with intact activating genes. Plasmids containing clones HSV-1 or HSV-2 immediate-early (IE) genes stimulated by up to 100-fold the expression from recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the DE promoter/regulatory region from the genes for an HSV-2 38,000-molecular-weight (38K) protein and the HSV-1 thymidine kinase. This activation was specific to hybrid genes containing DE regulatory regions since no significant increases in expression were observed in cotransfection experiments with the CAT gene without any promoter region or under the control of a number of other regulatory regions, including an HSV-1 1E regulatory region, the complete or enhancerless early regulatory region of simian virus 40, and an inducible cellular promoter/regulatory region. By using a variety of cotransfected plasmids containing individual or different combinations of HSV-1 or HSV-2 IE genes, we show that of the five known IE genes, two, those coding for the 175K and 110K polypeptides, each possessed the ability to stimulate expression from both DE promoters. Cleavage of the input plasmids within the known coding regions for the 175K and 110K proteins abolished stimulation of DE/CAT gene expression, whereas cleavage outside the coding regions had no effect on stimulation. We conclude that stimulation of CAT expression occurred exclusively by a transactivation mechanism in which the products encoded by these IE genes acted on the DE hybrid constructs at the transcription level. No transcriptional stimulatory function was demonstrated for the IE 68K and 63K proteins, although our results indicate that the IE 12K protein may augment the DE stimulatory activity of the 175K and 110K proteins.

AB - We reconstructed the regulated induction of delayed-early (DE) transcription that occurs during herpes simplex virus (HSV) infection by using a transient expression system in which recombinant target genes were cotransfected into Vero cells together with intact activating genes. Plasmids containing clones HSV-1 or HSV-2 immediate-early (IE) genes stimulated by up to 100-fold the expression from recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the DE promoter/regulatory region from the genes for an HSV-2 38,000-molecular-weight (38K) protein and the HSV-1 thymidine kinase. This activation was specific to hybrid genes containing DE regulatory regions since no significant increases in expression were observed in cotransfection experiments with the CAT gene without any promoter region or under the control of a number of other regulatory regions, including an HSV-1 1E regulatory region, the complete or enhancerless early regulatory region of simian virus 40, and an inducible cellular promoter/regulatory region. By using a variety of cotransfected plasmids containing individual or different combinations of HSV-1 or HSV-2 IE genes, we show that of the five known IE genes, two, those coding for the 175K and 110K polypeptides, each possessed the ability to stimulate expression from both DE promoters. Cleavage of the input plasmids within the known coding regions for the 175K and 110K proteins abolished stimulation of DE/CAT gene expression, whereas cleavage outside the coding regions had no effect on stimulation. We conclude that stimulation of CAT expression occurred exclusively by a transactivation mechanism in which the products encoded by these IE genes acted on the DE hybrid constructs at the transcription level. No transcriptional stimulatory function was demonstrated for the IE 68K and 63K proteins, although our results indicate that the IE 12K protein may augment the DE stimulatory activity of the 175K and 110K proteins.

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