Replication-associated activities of purified human papillomavirus type 11 E1 helicase.
Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.
Rocque
W J
WJ
Department of Molecular Sciences, Department of Molecular Biochemistry, Department of Virology, Glaxo Wellcome Inc., Five Moore Drive, Research Triangle Park, North Carolina 27709, USA.
Porter
D J
DJ
Barnes
J A
JA
Dixon
E P
EP
Lobe
D C
DC
Su
J L
JL
Willard
D H
DH
Gaillard
R
R
Condreay
J P
JP
Clay
W C
WC
Hoffman
C R
CR
Overton
L K
LK
Pahel
G
G
Kost
T A
TA
Phelps
W C
WC
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002942:Circular Dichroism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000821:virology; D051379:Mice; D043583:Nucleoside-Triphosphatase; D027383:Papillomaviridae / Q000737:chemistry*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; D014764:Viral Proteins; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases