Involvement of a high-mobility-group protein in the transcriptional activity of herpes simplex virus latency-active promoter 2.
Latency-active promoter 2 (LAP 2) is a TATA-less promoter in herpes simplex virus type 1 (HSV-1) that can express genes during viral latency. Four regions of LAP2 are protected from DNase I digestion in vitro by either HeLa cell nuclear extracts or purified Sp1. Transient gene expression assays of LAP2 substitution mutants demonstrate that two of the regions protected by Sp1 and three other regions protected by nuclear extract are important for promoter function. The mutation causing the most significant reduction in expression alters a stretch of 23 thymidine residues (T23) that binds a protein with several properties common to high-mobility-group (HMG) proteins. The T23 binding activity is heat stable, can be inhibited by poly(dA-dT).poly(dA-dT), and is inhibited by minor-groove-binding drugs. Antiserum directed against HMG I(Y) blocked the formation of one of the DNA-protein complexes on the T23 oligonucleotide, suggesting that a protein antigenically related to HMG I(Y) binds to LAP2 in vitro. Direct evidence of HMG I(Y) involvement in LAP2 function is provided by the findings that recombinant HMG I(Y) protein facilitates Sp1 binding to LAP2 in mobility shift assays and that antisense HMG I(Y) RNA specifically inhibits LAP2 function in vivo. These results suggest that DNA structure may be an important determinant of the activity of a promoter that is capable of escaping the global shutoff of transcription that occurs during viral latency.
French
S W
SW
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Schmidt
M C
MC
Glorioso
J C
JC
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D018983:DNA Footprinting; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry; D003850:Deoxyribonuclease I; D017930:Genes, Reporter; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014709:Vero Cells
D017931:DNA Primers; D004279:DNA, Viral; D006609:High Mobility Group Proteins; D016329:Sp1 Transcription Factor; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I