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PMID: 2845144 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein.

Journal of virology ·Vol. 62 ·No. 11 ·1988-11-00 ·Pages 4307-20

Roberts MS, Boundy A, O'Hare P, Pizzorno MC, Ciufo DM, Hayward GS

Abstract

In transient-expression assays, the IE175 (alpha 4) promoter region of herpes simple virus is down-regulated after cotransfection with DNA encoding its own protein product (IE175 or ICP4). The inhibition by IE175 proved to be highly specific for its own promoter region and did not act on either the herpes simplex virus type 1 IE110 (alpha 0) or human cytomegalovirus major immediate-early promoters. Furthermore, the inhibition was still exhibited by IE175 effector plasmids driven by strong heterologous promoters and therefore must be a direct autoregulatory response that cannot be explained by promoter competition effects. In gel mobility retardation assays with infected-cell nuclear extracts, a prominent and specific DNA-protein complex was formed with DNA fragments containing sequences from -108 to +30 in the IE175 promoter region. This activity was not present in mock-infected samples. Even stronger binding occurred with a fragment containing sequences from -128 to +120 in the IE110 promoter, but this second locus was not associated with any detectable response phenotype in cotransfection assays. Supershift experiments with an anti-IE175 monoclonal antibody confirmed the presence of the IE175 protein in both DNA-protein complexes. In the IE175 promoter, specific binding correlated closely with the presence of an intact autoregulatory signal near the cap site as judged by the loss of both activities in a 3'-deleted promoter fragment lacking sequences from -7 to +30. Insertion of a cloned 30-mer synthetic oligonucleotide sequence from positions -8 to +18 in IE175 restored both IE175 binding activity and the down-regulation phenotype. Direct shift-up assays with a similar 30-base-pair (bp) oligonucleotide containing 21 bp from positions -75 to -55 of IE110 (which encompasses a consensus ATCGTC motif) also produced a specific DNA-protein complex containing the IE175 protein. This ATCGTC motif proved to be a necessary component of both the IE110 and IE175 binding sites, but was insufficient on its own for complex formation. Finally, deletion of 2 bp from positions -3 and -4 within the ATCGTC sequence in the IE175 cap site region abolished both binding activity and the IE175-dependent autoregulation phenotype.

MeSH Terms
Animals Base Sequence Binding Sites Cell Extracts Chloramphenicol O-Acetyltransferase Cloning, Molecular DNA-Binding Proteins/metabolism,physiology Electrophoresis, Polyacrylamide Gel Gene Expression Regulation Genes, Viral Homeostasis Immediate-Early Proteins Oligonucleotide Probes/chemical synthesis Plasmids Promoter Regions, Genetic Restriction Mapping Simplexvirus/genetics Transcription Factors/metabolism,physiology Transfection Vero Cells Viral Proteins/metabolism,physiology
Chemicals
Cell Extracts DNA-Binding Proteins Immediate-Early Proteins Oligonucleotide Probes Transcription Factors Viral Proteins herpes simplex virus, type 1 protein ICP4 Chloramphenicol O-Acetyltransferase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Roberts M S
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Boundy A
O'Hare P
Pizzorno M C
Ciufo D M
Hayward G S
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.62.11.4307-4320.1988
Published
1988-11-00
Pages
4307-20
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC253866
Subset
IM
Grants
NCI NIH HHS · R01 CA22130 · United States
NCI NIH HHS · R01 CA28473 · United States
External Links
PubMed source DOI: 10.1128/JVI.62.11.4307-4320.1988 PMC Full Text View journal details
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