Contribution of bovine papillomavirus type 1 E1 protein residue 48 to replication function.
The E1 protein of bovine papillomavirus type 1 (BPV-1) is the origin recognition protein and is essential for the initiation of viral DNA replication. We reported previously that there is a conserved motif between residues 25 and 60 of all papillomavirus E1 proteins that resembles a casein kinase II (CKII) phosphorylation site. The corresponding serine in BPV-1, serine-48, is an efficient substrate for CKII in vitro. To examine the functional role of this potential phosphorylation site, three amino acid substitutions were constructed at serine-48. Conversion of serine-48 to a glycine (S48G) resulted in a BPV-1 genome that was unable to replicate and had reduced transformation capacity. The S48G E1 protein also failed to support replication of a BPV-1 origin-containing plasmid when expressed from a heterologous vector rather than the viral genome, indicating a direct replication defect. In contrast, conversion of serine-48 to acidic residues (S48D or S48E), which mimic the charge and structure of phosphoserine, maintained the wild-type replication phenotype. These mutational results are consistent with a replication requirement for a negative charge at serine-48, presumably supplied by in vivo phosphorylation. The mechanistic basis for the negative charge requirement was examined by testing several activities of the S48G mutant E1 protein in vivo using yeast one- and two-hybrid systems. No gross defect was observed for stability, origin binding or interaction with E2 or for E1-E1 interaction, although subtle defects in these activities would not likely be detected. Overall, the results suggest that important phosphoregulatory control of E1 replication function is mediated through the N-terminal region of this protein.
McShan
Gina D
GD
Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA1.
Wilson
Van G
VG
Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA1.
D000595:Amino Acid Sequence; D000818:Animals; D052688:Bovine papillomavirus 1 / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D017124:Conserved Sequence; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
D004268:DNA-Binding Proteins; C069814:E1 protein, Bovine papillomavirus; D014764:Viral Proteins