Identification of Domains of the HPV11 E1 Protein Required for DNA Replication in Vitro

Amin, Anthony A.; Titolo, Steve; Pelletier, Alex; Fink, D; Cordingley, Michael G.; Archambault, Jacques

doi:10.1006/viro.2000.0328

Cited by 41 publications

(58 citation statements)

References 50 publications

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“…A similar truncation of HPV33 E1 was reported to be expressed at higher levels than the full-length protein in E. coli (61). Furthermore, we have shown previously that this truncated E1, when made by in vitro translation, retains the ability to support cell-free DNA replication (25). These three E1 proteins were expressed as fusions with an N-terminal 6-histidine tag to facilitate their rapid purification by metal affinity chromatography and hence minimize losses of activity over the course of the procedure.…”

Section: Expression and Purification Of Hpv11 And Hpv6 E1-wesupporting

confidence: 57%

“…For BPV, it has been demonstrated that ATP hydrolysis is required for displacement of E2 from the origin during assembly of E1 multimeric complexes (17). Either concurrent with or after formation of E1 oligomers, the host polymerase ␣ primase (21)(22)(23)(24)(25) and possibly also replication protein A (22,26) bind to E1, and a replication complex is formed together with additional host factors.…”

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

“…We have recently shown that amino acids 166 -649 of E1 efficiently support DNA replication in a cell-free system and thus must encode for all necessary binding and enzymatic activities required in vitro (25). In vivo, additional sequences spanning amino acids 82-128 (HPV11 E1) are required; these contain an extended nuclear localization sequence (27,28) as well as a cyclin-binding motif and cyclin-dependent kinase phosphorylation sites (29 -31).…”

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

“…A minimal DNA binding/origin recognition sequence was mapped to approximately amino acids 140 -300 for BPV E1 (32)(33)(34)(35) (amino acids 185-345 for HPV11 E1), although the entire C terminus of the protein (amino acids 186 -649) is required to observe stable binding of HPV11 E1 to the origin (20,36). An HPV11 E1 truncation consisting of the C-terminal amino acids 353-649 retains the ability to bind to E2 or the p70 subunit of polymerase ␣ primase and to oligomerize into hexamers (19,20,25). Within this C-terminal fragment of E1 are four conserved re-gions termed A, B, C, and D (see Fig.…”

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

“…The locations of conserved motifs a, b, and c that are common to members of superfamily 3 NTP-binding proteins (38) are indicated by arrows. Indicated below E1 are truncated constructs of the protein that we have found in previous studies to be sufficient for catalyzing DNA replication in a cell-free system, for stable binding to the origin, for interaction with the transactivation domain of E2 (19) or the p70 subunit of polymerase ␣ primase (25), or for oligomerization of the protein (20). The position of a minimal E1-E1 interaction domain mapped between amino acids 353 and 416 using the yeast two-hybrid system (20) is indicated by a hatched box.…”

Section: Construction Of Recombinant Baculoviruses-mentioning

confidence: 99%

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Characterization of Recombinant HPV6 and 11 E1 Helicases

White

Pelletier

Brault

et al. 2001

Journal of Biological Chemistry

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To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K m (ATP) of 12 M for HPV6 E1 and 6 M for HPV11 E1. Interestingly, the value of K m (ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.The papillomaviruses (PVs) 1 are small, nonenveloped DNA viruses that infect and replicate in the cutaneous or mucosal epithelia of humans and other mammals. There are over 100 types of human papillomavirus (HPV), which cause conditions ranging from plantar warts (HPV1) and genital warts (HPV6 and -11) to cervical cancer (HPV16, -18, and -31). HPV6 and -11 are also responsible for laryngeal papillomatosis, a rare but very serious infection of the respiratory tract (1). Antiviral agents capable of specifically inhibiting PV replication could play an important role in the treatment of these diseases, but none exist at this time.Despite their host and tissue specificity, all of the PV types share a common genomic organization. The closed, circular genome of ϳ8000 base pairs codes for only 10 proteins: eight early proteins termed E1-E8 and two late proteins, L1 and L2, that make up the viral coat (2). E1 and E2 are the only viral proteins required for HPV DNA replication (3, 4). E2 is a sequence-specific DNA-binding protein that serves to regulate both transcription and DNA replication. E1, a DNA helicase, is the only PV protein that possesses enzymatic activity (5) and is also the most highly conserved of the PV proteins. For these reasons, E1 has been considered as the most attractive molecular target for the development of antiviral agents (6).During the initiation of PV DNA replication, E2 serves as a specificity factor to enhance binding of E1 monomers to the origin, which by themselves bind to double-stranded DNA with little sequence specificity (4, 7-11). E2 dimers bind with high specificity to DNA sequences within the origin and recruit to it E1 monomers, through direct protein-protein interactions (12, 13). Upon binding to DNA, E1 monomers assemble into hexamers (14, 15), but the E1-E2 interaction is not maintained (16,17). Thus, the role of E2 is to catalyze the assembly of hexameric E1 complexes specifically at the origin. ATP hydrolysis is also required for E1 hexamers to distort the orig...

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Section: Expression and Purification Of Hpv11 And Hpv6 E1-wesupporting

confidence: 57%

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

Section: Construction Of Recombinant Baculoviruses-mentioning

confidence: 99%

See 3 more Smart Citations

Characterization of Recombinant HPV6 and 11 E1 Helicases

White

Pelletier

Brault

et al. 2001

Journal of Biological Chemistry

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Identification of a novel human papillomavirus type 16 E1 gene variant with potentially reduced oncogenicity

Sabol

Matovina

Gašperov

et al. 2008

Journal of Medical Virology

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The human papillomavirus (HPV) 16 genome has been studied extensively, although no study has focused on the E1 gene that is implicated in viral DNA replication. After analyzing the E1 region of HPV 16 genomes in 429 cervical samples, 11.2% were found to contain a 63 nucleotides duplication in this region. Sequence analysis of the E6 and the E7 regions has shown that all samples containing this duplication were related to E6‐G350 variant of the HPV 16 (Chi square test, P = 0.0012). A comparison of cervical lesion severity of the examinees having regular or variant E1 genes has shown that the variant group had a significantly (Fischer's exact test, P = 0.0401) lower percentage of high grade disease cases, suggesting that this particular duplication might reduce the oncogenic potential of HPV 16, and also might clarify the differences of E6‐G350 variant oncogenicity observed in European populations. Albeit, a decreased incidence of high grade cervical lesions can be linked to the prevalence of multiple HPV infection, the additional decrease of those cases with the variant E1 gene versus those without (10.5% and 18.6%, respectively) can only be ascribed to the effect of this particular HPV variant. Further research is needed to clarify the biology of these HPV 16 E1 variants. J. Med. Virol. 80:2134–2140, 2008. © 2008 Wiley‐Liss, Inc.

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A Quantitative and High-Throughput Assay of Human Papillomavirus DNA Replication

Gagnon

Fradet-Turcotte

Archambault

2014

Methods in Molecular Biology

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Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

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Identification of Domains of the HPV11 E1 Protein Required for DNA Replication in Vitro

Cited by 41 publications

References 50 publications

Characterization of Recombinant HPV6 and 11 E1 Helicases

Characterization of Recombinant HPV6 and 11 E1 Helicases

Identification of a novel human papillomavirus type 16 E1 gene variant with potentially reduced oncogenicity

A Quantitative and High-Throughput Assay of Human Papillomavirus DNA Replication

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