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1.
J Virol ; 73(6): 4670-7, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10233926

ABSTRACT

The bovine papillomavirus type 1 (BPV-1) E2 protein is the master regulator of papillomavirus replication and transcription. We have raised a panel of monoclonal antibodies (MAbs) against the BPV-1 E2 protein and used them to probe the structure and function of the protein. Five MAbs reacted with linear epitopes, and four MAbs recognized conformation-dependent epitopes which mapped within the C-terminal DNA-binding and dimerization domain. MAb 1E2 was able to recognize the replication- and transactivation-defective but not the competent conformation of the transactivation domain of the E2 protein. MAb 5H4 prevented efficiently the formation of E2-DNA as well as E2-dependent E1-E2-origin complexes and also dissociated preformed complexes in a concentration-dependent manner. Cotransfection of several MAbs with the BPV-1 minimal origin plasmid pUCAlu into CHO4.15 cells resulted in a dose-dependent inhibition of replication. Inhibition of replication by MAb 5H4 and the Fab' fragment of 5H4 correlated with their ability to dissociate the E2 protein from the DNA. MAb 3F12 and MAbs 1H10 and 1E4, directed against the hinge region, were also capable of inhibiting BPV-1 origin replication in CHO4.15 cells. However, the Fab' fragments of 1H10 and 3F12 had no effect in the transient replication assay. These data suggest that MAbs directed against the hinge region sterically hinder the inter- or intramolecular interactions required for the replication activity of the E2 protein.


Subject(s)
Antibodies, Monoclonal/pharmacology , Bovine papillomavirus 1/physiology , DNA Replication , DNA-Binding Proteins/physiology , Viral Proteins/physiology , Virus Replication , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , CHO Cells , COS Cells , Cricetinae , DNA-Binding Proteins/immunology , Epitope Mapping , Female , Immunoglobulin Fab Fragments/pharmacology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Viral Proteins/immunology
2.
J Virol ; 70(9): 6169-79, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8709243

ABSTRACT

A set of E2 proteins with mutations in the amino-terminal transactivation domain was made by a scheme called clustered charged-to-alanine scan. These mutant E2 proteins were tested for expression, stability, and compartmentalization in cells and for sequence-specific DNA binding, as well as in functional assays for transcriptional and replicational activation. We identified four groups of mutants. First, mutants K111A, K112A, and E176A were unable to activate replication and transcription because of oligomerization-induced retention of oligomers in the cytoplasm. Second, although fractions of the mutant proteins E74A and D143A/ R172C existed in the oligomeric form, they were localized in the nucleus. Certain fractions of these proteins existed as a dimer able to form a specific complex and activate replication; however, these proteins were inactive in transcriptional activation. Third, mutants R37A and D122A were localized in the nucleus, existed in the dimeric form, supported replication efficiently, and were severely crippled in transcriptional activation. The fourth group of mutants did not differ considerably from the wild-type protein. The activation of transcription by the wild type as well as mutant E2 proteins was dependent on the concentration of input E2 expression vector DNA and had a bell-like shape. We suggest that the reduction of transcriptional activation at higher E2 concentrations, the self-squelching activity, is caused by oligomerization of the E2 transactivator and is one of the mechanisms for the regulation of E2 activity. Our results also show that transcriptional and replicational activation activities are encoded by different determinants in the E2 protein.


Subject(s)
Bovine papillomavirus 1/physiology , DNA Replication , DNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Viral Proteins/metabolism , Virus Replication , Alanine , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Bovine papillomavirus 1/genetics , CHO Cells , Cattle , Cell Line , Chlorocebus aethiops , Codon , Cricetinae , DNA Probes , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Point Mutation , Promoter Regions, Genetic , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , Viral Proteins/genetics , Viral Proteins/isolation & purification
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