A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator

Robert T. Sarisky, Zhigang Gao, Paul M. Lieberman, Elizabeth D. Fixman, Gary Selwyn Hayward, S. Diane Hayward

Research output: Contribution to journalArticle

Abstract

The Zta transactivator is crucial for both Epstein-Barr virus (EBV) lytic gene expression and lytic DNA replication. We have used a cotransfection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication activity. Deletion of Zta aa 25 to 86, which are critical for transcriptional activation of ori-Lyt, or aa 93 to 141 did not adversely affect replication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (Δ2- 25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (Δ2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication competency, while deletion of codons 13 to 19 (Δ13-19) or mutation at codons 12 and 13 (m12/13) impaired Zta replication function. Each of the replication-negative Zta variants was capable of transactivating expression from both BHLF1 promoter-chloramphenicol acetyltransferase constructions and the BMRF1 promoter on endogenous EBV genomes in Raji cells with efficiency comparable to that of the wild-type polypeptide. Thus, a replication contribution of Zta was functionally separable from its transactivation activity and was supplied by the N- terminal region encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the replication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region. A chimeric Rta-EBNA-2 transactivation domain fusion, which retains the DNA-binding and transactivation properties associated with wild-type Rta, failed to rescue replication-deficient Zta. Our data suggest that Rta may act as an ancillary, replication factor in EBV ori-Lyt DNA synthesis by stabilizing Zta-replisome interactions.

Original languageEnglish (US)
Pages (from-to)8340-8347
Number of pages8
JournalJournal of Virology
Volume70
Issue number12
StatePublished - Dec 1996

Fingerprint

Human herpesvirus 4
Trans-Activators
transcriptional activation
Human Herpesvirus 4
Transcriptional Activation
amino acid deletion
codons
Amino Acids
amino acids
Codon
mutation
Mutation
promoter regions
chloramphenicol acetyltransferase
Aminoacylation
DNA
assays
DNA replication
Chloramphenicol O-Acetyltransferase
polypeptides

ASJC Scopus subject areas

  • Immunology

Cite this

Sarisky, R. T., Gao, Z., Lieberman, P. M., Fixman, E. D., Hayward, G. S., & Hayward, S. D. (1996). A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator. Journal of Virology, 70(12), 8340-8347.

A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator. / Sarisky, Robert T.; Gao, Zhigang; Lieberman, Paul M.; Fixman, Elizabeth D.; Hayward, Gary Selwyn; Hayward, S. Diane.

In: Journal of Virology, Vol. 70, No. 12, 12.1996, p. 8340-8347.

Research output: Contribution to journalArticle

Sarisky, RT, Gao, Z, Lieberman, PM, Fixman, ED, Hayward, GS & Hayward, SD 1996, 'A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator', Journal of Virology, vol. 70, no. 12, pp. 8340-8347.
Sarisky, Robert T. ; Gao, Zhigang ; Lieberman, Paul M. ; Fixman, Elizabeth D. ; Hayward, Gary Selwyn ; Hayward, S. Diane. / A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator. In: Journal of Virology. 1996 ; Vol. 70, No. 12. pp. 8340-8347.
@article{ea9b41f019414fe69888754015c8185a,
title = "A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator",
abstract = "The Zta transactivator is crucial for both Epstein-Barr virus (EBV) lytic gene expression and lytic DNA replication. We have used a cotransfection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication activity. Deletion of Zta aa 25 to 86, which are critical for transcriptional activation of ori-Lyt, or aa 93 to 141 did not adversely affect replication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (Δ2- 25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (Δ2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication competency, while deletion of codons 13 to 19 (Δ13-19) or mutation at codons 12 and 13 (m12/13) impaired Zta replication function. Each of the replication-negative Zta variants was capable of transactivating expression from both BHLF1 promoter-chloramphenicol acetyltransferase constructions and the BMRF1 promoter on endogenous EBV genomes in Raji cells with efficiency comparable to that of the wild-type polypeptide. Thus, a replication contribution of Zta was functionally separable from its transactivation activity and was supplied by the N- terminal region encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the replication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region. A chimeric Rta-EBNA-2 transactivation domain fusion, which retains the DNA-binding and transactivation properties associated with wild-type Rta, failed to rescue replication-deficient Zta. Our data suggest that Rta may act as an ancillary, replication factor in EBV ori-Lyt DNA synthesis by stabilizing Zta-replisome interactions.",
author = "Sarisky, {Robert T.} and Zhigang Gao and Lieberman, {Paul M.} and Fixman, {Elizabeth D.} and Hayward, {Gary Selwyn} and Hayward, {S. Diane}",
year = "1996",
month = "12",
language = "English (US)",
volume = "70",
pages = "8340--8347",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "12",

}

TY - JOUR

T1 - A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator

AU - Sarisky, Robert T.

AU - Gao, Zhigang

AU - Lieberman, Paul M.

AU - Fixman, Elizabeth D.

AU - Hayward, Gary Selwyn

AU - Hayward, S. Diane

PY - 1996/12

Y1 - 1996/12

N2 - The Zta transactivator is crucial for both Epstein-Barr virus (EBV) lytic gene expression and lytic DNA replication. We have used a cotransfection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication activity. Deletion of Zta aa 25 to 86, which are critical for transcriptional activation of ori-Lyt, or aa 93 to 141 did not adversely affect replication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (Δ2- 25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (Δ2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication competency, while deletion of codons 13 to 19 (Δ13-19) or mutation at codons 12 and 13 (m12/13) impaired Zta replication function. Each of the replication-negative Zta variants was capable of transactivating expression from both BHLF1 promoter-chloramphenicol acetyltransferase constructions and the BMRF1 promoter on endogenous EBV genomes in Raji cells with efficiency comparable to that of the wild-type polypeptide. Thus, a replication contribution of Zta was functionally separable from its transactivation activity and was supplied by the N- terminal region encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the replication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region. A chimeric Rta-EBNA-2 transactivation domain fusion, which retains the DNA-binding and transactivation properties associated with wild-type Rta, failed to rescue replication-deficient Zta. Our data suggest that Rta may act as an ancillary, replication factor in EBV ori-Lyt DNA synthesis by stabilizing Zta-replisome interactions.

AB - The Zta transactivator is crucial for both Epstein-Barr virus (EBV) lytic gene expression and lytic DNA replication. We have used a cotransfection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication activity. Deletion of Zta aa 25 to 86, which are critical for transcriptional activation of ori-Lyt, or aa 93 to 141 did not adversely affect replication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (Δ2- 25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (Δ2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication competency, while deletion of codons 13 to 19 (Δ13-19) or mutation at codons 12 and 13 (m12/13) impaired Zta replication function. Each of the replication-negative Zta variants was capable of transactivating expression from both BHLF1 promoter-chloramphenicol acetyltransferase constructions and the BMRF1 promoter on endogenous EBV genomes in Raji cells with efficiency comparable to that of the wild-type polypeptide. Thus, a replication contribution of Zta was functionally separable from its transactivation activity and was supplied by the N- terminal region encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the replication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region. A chimeric Rta-EBNA-2 transactivation domain fusion, which retains the DNA-binding and transactivation properties associated with wild-type Rta, failed to rescue replication-deficient Zta. Our data suggest that Rta may act as an ancillary, replication factor in EBV ori-Lyt DNA synthesis by stabilizing Zta-replisome interactions.

UR - http://www.scopus.com/inward/record.url?scp=0029861946&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029861946&partnerID=8YFLogxK

M3 - Article

C2 - 8970953

AN - SCOPUS:0029861946

VL - 70

SP - 8340

EP - 8347

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 12

ER -