Summary auto-generated
The E1 protein of bovine papillomavirus type 1 (BPV-1) is essential for viral DNA replication and is known to be phosphorylated in vivo. This study examined predicted amino acid sequences of E1 proteins from multiple papillomavirus species and identified a conserved motif between amino acids 25-60 that resembles a phosphorylation consensus site for casein kinase II (CKII). The researchers used in vitro biochemical assays with purified CKII and bacterially expressed E1 fusion proteins to demonstrate that BPV-1 E1 is a substrate for CKII phosphorylation. Through phosphoamino acid analysis, they showed that phosphorylation occurred exclusively on serine residues. Using a series of truncated E1 fragments and site-directed mutagenesis, they mapped the primary CKII phosphorylation site to serine-48 (Ser-48) in the N-terminal region. Mutation of Ser-48 to glycine significantly reduced but did not eliminate E1 phosphorylation, indicating the presence of additional minor CKII phosphorylation sites. The strong conservation of this CKII recognition motif across diverse papillomavirus E1 proteins suggests biological significance for this post-translational modification in regulating viral replication and host cell cycle functions.
Key findings
- BPV-1 E1 protein is phosphorylated in vitro by casein kinase II at a conserved N-terminal motif containing serine-48
- The CKII phosphorylation site (Ser-X-X-Asp/Glu motif) at amino acids 25-60 is strongly conserved across all examined papillomavirus E1 proteins
- Phosphorylation occurs exclusively on serine residues, not threonine, and is specific to the N-terminal region encompassing amino acids 1-75
- Site-directed mutagenesis of Ser-48 significantly reduces but does not eliminate E1 phosphorylation, indicating additional minor CKII sites exist
- E1 Ser-48 mutations show defective viral replication in vivo, suggesting this phosphorylation site has functional importance for BPV-1 biology
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Abstract
The E1 protein of bovine papillomavirus type 1 (BPV-1) is a phosphoprotein which specifically binds and unwinds the virus replication origin by ATP-dependent helicase activity. The El protein has been shown to be multiply phosphorylated in vivo, although the sites of modification are incompletely mapped. Examination of the predicted amino acid sequence of all available E1 proteins revealed strong conservation between amino acids 25 and 60 of a motif consisting of a serine residue followed by a stretch of acidic residues. This conserved motif resembled a phosphorylation consensus site for the ubiquitous cellular kinase casein kinase II (CKII). Biochemical and mutational analysis demonstrated that the BPV- 1 E1 protein is an in vitro substrate for CKII at the serine within this conserved motif.