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Van G Wilson

Publications and source records attributed to Van G Wilson.

5 recordsLinked to original sources
PubMed2002

Hydrophobic residue contributions to sequence-specific DNA binding by the bovine papillomavirus helicase E1.

Previously, mutational analyses of the DNA binding domain of the bovine papillomavirus E1 protein (E1DBD) identified several hydrophobic residues that are critical for DNA binding activity (M. West, D. Flanery, K. Woytek, D. Rangasamy, and V. G. Wilson, 2001, J. Virol. 75, 11948-11960). Hydrophobic interactions of nonpolar amino acid side chains can contribute to the function of DNA binding proteins through both conformational effects and direct interaction with nucleotides. To further investigate the role of hydrophobic residues in E1DBD function, a more extensive site-directed mutational analysis of hydrophobic amino acids was conducted. Alanine substitutions were made at residues V196, F197, F217, F, 237, V246, L249, and F276, and the mutants were tested for DNA binding activity in vitro and in vivo. The E1 F237A and F276A mutants were completely defective for site-specific DNA binding, while the other mutants retained partial to full wild-type binding activity. Consistent with their DNA binding defect, the F237A and F276A mutants were severely impaired for the ability to support transient in vivo replication of an origin plasmid. Combined with our previous study, five critical hydrophobic residues have been identified: F175, V193, F237, V246, and F276. These five residues localize to two internal clusters in the E1DBD structure designated hydrophobic clusters A (HCA; includes F175, V193, and F276) and B (HCB; includes F237 and V246). Amino acid side chains from residues in HCA and HCB have little surface accessibility and it is unlikely that they are involved in direct contact with DNA. HCA is distal to the DNA binding surface and presumably contributes to global conformational organization of the E1DBD. HCB is positioned beneath the DNA contact surface and we propose that it serves as an anchor or platform device to stabilize the DNA-binding element. A comparable hydrophobic cluster is present in the corresponding position in the T antigen DBD and likely serves a similar function.

Alanine↗
PubMed2002

Papillomavirus E1 proteins: form, function, and features.

The E1 proteins are the essential origin recognition proteins for papillomavirus (PV) replication. E1 proteins bind to specific DNA elements in the viral origin of replication and assemble into hexameric helicases with the aid of a second viral protein, E2. The resultant helicase complex initiates origin DNA unwinding to provide the template for subsequent syntheses of progeny DNA. In addition to ATP-dependent helicase activity, E1 proteins interact with and recruit several host cell replication proteins to viral origin, including DNA polymerase alpha and RPA. This review will compare the basic structures and features of the human (HPV) and bovine (BPV1) papillomaviruses with an emphasis on mechanisms of replication function.

Adenosine Triphosphatases↗
PubMed2001

Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1-E2 interaction.

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1-E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1-311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315-605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N-E2TAD interaction is not essential for replication function and is probably involved in some other E1-E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.

Animals↗
PubMed2000

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.

Amino Acid Sequence↗