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Robert D. Burk - One of the best experts on this subject based on the ideXlab platform.
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Transient replication analysis of the MfPV wild-type and mutant genomes in the U2OS cell line.
2019Co-Authors: Eva-maria Tombak, Andres Männik, Robert D. Burk, Roger Le Grand, Ene Ustav, Mart UstavAbstract:U2OS cells were transfected with 4 μg of wild-type (wt) or mutant minicircle genomes of MfPV5 (A), MfPV8 (B), and MfPV1 (C) defective in E1 (E1-), E2 (E2-) and/or E8^E2C (E8-) expression. For the complementation assay, the cells were cotransfected with E1- and E2-defective genomes (4 μg each). Episomal DNA was extracted via Hirt lysis at the indicated time points (days) and subjected to Southern blotting. Prior to analysis, the samples were treated with DpnI and specific endonucleases (Thermo Scientific). (A) In the case of MfPV5, the E8-E1- and E8-E2- double mutant genomes were used. MfPV5 wt (lines 1–3), E8-E1- (lines 4–6) and E8-E2- (lines 7–9) samples were digested with the linearizing enzyme BamHI, while E8-E1- plus E8-E2- samples (lines 10–12) were double digested with BamHI and SacI (only cuts the E2 mutant genome) to facilitate distinction between cotransfected plasmids. (B) MfPV8 wt (lines 1–3), E1- (lines 4–6) and E2- (lines 7–9) samples were digested with the linearizing enzyme BglI, while E1- plus E2- samples (lines 10–12) were double digested with BglI and BglII (only cuts the E2 mutant genome). (C) In the case of MfPV1, the E8-E1- and E8-E2- double mutant genomes were also used. MfPV1 wt (lines 1–3), E8-E1- (lines 4–6) and E8-E2- (lines 7–9) samples were digested with the linearizing enzyme Eam1105I, while E8-E1- plus E8-E2- samples (lines 10–12) were double digested with Eam1105I and HindIII (only cuts the E1 mutant genome). DpnI-treated, linear and double digested marker plasmids for MfPV5, MfPV8 or MfPV1 are shown in lanes 16 to 18. In the case of complementation assay, linear viral genome is indicated with arrows, double digested genome fragments with arrowheads. Abbreviations: E1-, E1 mutant genomes; E2-, E2 mutant genomes.
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human papillomavirus hpv types 101 and 103 isolated from cervicovaginal cells lack an e6 open reading frame orf and are related to gamma papillomaviruses
Virology, 2007Co-Authors: Zigui Chen, Rolando Herrero, Rob Desalle, Mark Schiffman, Robert D. BurkAbstract:Complete genomes of HPV101 and HPV103 were PCR amplified and cloned from cervicovaginal cells of a 34-year-old female with cervical intraepithelial neoplasia grade 3 (CIN 3) and a 30-year-old female with a normal Pap test, respectively. HPV101 and HPV103 contain 4 early genes (E7, E1, E2, and E4) and 2 late genes (L2 and L1), but both lack the canonical E6 ORF. Pairwise alignment similarity of the L1 ORF nucleotide sequences of HPV101 and HPV103 indicated that they are at least 30% dissimilar to each other and all known PVs. However, similarities of the other ORFs (E7, E1, E2, and L2) indicated that HPV101 and HPV103 are most related to each other. Phylogenetic analyses revealed that these two types form a monophyletic clade, clustering together with the gamma- and pi-PV groups. These data demonstrated that HPV genomes closely related to papillomaviruses identified from cutaneous epithelia can be isolated from the genital mucosal region. Moreover, this is the first report of HPVs lacking an E6 ORF and phylogenetic evidence suggests this occurred subsequent to their emergence from the gamma-/pi-PVs.
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Diversifying selection in human papillomavirus type 16 lineages based on complete genome analyses.
Journal of virology, 2005Co-Authors: Zigui Chen, Masanori Terai, Rolando Herrero, Rob Desalle, Robert D. BurkAbstract:Papillomaviruses are a heterogeneous group of DNA viruses with closed circular double-stranded DNA genomes of about 8 kb in size that contain three general regions. An upstream regulatory region (URR) contains sequences that control transcription and replication, an early region contains genes (e.g., E6, E7, E1, E2, E4, and E5) involved primarily in enzymatic activities, and a structural region produces the L1 capsid protein and L2, which facilitates packaging of the viral DNA. Human papillomaviruses (HPVs) are classified by the sequence similarity of their genomes. A cloned HPV genome whose L1 open reading frame (ORF) displays less than 90% similarity to previously designated types is defined as a novel type. To date, more than 90 different genotypes of the HPV have been fully characterized (21). Intratypic variants and subtypes are defined as HPVs that vary by less than 10% in their L1 DNA sequences (5, 21). HPVs are causally involved in the etiology of cervical cancer and its precursor lesions (16, 17, 43, 49). Of the high-risk HPV types associated with cervical cancer, HPV16 is the most prevalent and is found in approximately half of all cancers (7, 49). Numerous variants of HPV16 have been identified in different geographic locations and ethnic groups (35, 42, 53, 68). Although all HPV16 isolates are closely related, previous studies inferred five distinct phylogenetic branches among HPV16 variants: European (E), Asian (As), Asian-American (As-Am), African-1 (Af-1), and African-2 (Af-2), corresponding to the geographic locations from which the samples were obtained (12, 34). Subsequent studies by sequence analyses of theHPV16 variants in other genomic regions (e.g., E6, L2, and L1) expanded and complemented this phylogenetic hypothesis (69). Although HPV16 variants are an important focus of phylogenetic studies and the molecular variants of E2, L2, L1, the URR, and especially the E6 region have been described in detail previously (28), covariation among different ORFs belonging to the same lineage or isolate have not been studied in great detail. HPV16 variants have demonstrable differences in biological properties in vitro which may be responsible, in part, for differences in pathogenicity, carcinogenic risk, and perhaps immunogenicity (28). Furthermore, HPV16 variants are associated with different cervical cancer risks (6, 32, 65, 67). Although the diversifying selection in the HPV16 E6 and E7 oncogenes has been described recently (20), the evolutionary basis of the entire genome, coevolutionary mechanisms among different HPV16 genes, and their underlying biological significance remain unknown. Obtaining whole-genome sequences representative of the major HPV16 variants allowed us to determine with certainty nucleotide and amino acid sequence changes that are of potential evolutionary importance. Comparison of synonymous (silent; dS) and nonsynonymous (amino acid-changing; dN) substitution rates in protein-coding genes provides an important means for investigating the forces of molecular evolution (72). The nonsynonymous/synonymous rate ratio (ω = dN /dS) measures selective pressure at the protein level. Nonsynonymous rates that are significantly higher than synonymous rates are accepted as evidence for molecular adaptation (71, 72). This criterion has been used to identify a number of genes under adaptive molecular evolution (45). In this report, the complete nucleotide sequences of and codon variations within HPV16 genomes representing the five major lineages were determined. By examining the ratio of dN to dS substitutions per site, diversifying selection acting on all of the eight protein-coding regions was evaluated. In addition, complete genome sequences for these variants allow us to reconstruct their genealogical relationships with unprecedented precision.
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the mastomys natalensis papillomavirus nucleotide sequence genome organization and phylogenetic relationship of a rodent papillomavirus involved in tumorigenesis of cutaneous epithelia
Virology, 1994Co-Authors: Chiew Hoon Tan, Ruth Tachezy, Marc Van Ranst, Shih Yen Chan, Hansulrich Bernard, Robert D. BurkAbstract:Mastomys natalensis is a rodent of African origin afflicted with a very high incidence of skin tumors (keratoacanthomas and squamous carcinomas), which are associated with a papillomavirus, M. natalensis papillomavirus (MnPV). We have determined the genomic sequence of MnPV, which has a size of 7687 bp. The genomic organization is similar to that of other papillomaviruses, with open reading frames E6, E7, E1, E2, and E4 in the early and L2 and L1 in the late region. Due to an unusually large hinge region, the transcriptional activator E2 has a size of 542 amino acids rather than 400 to 460 amino acids, as in other papillomaviruses. An open reading frame E5 coding for a small hydrophobic membrane protein is missing, as is the case for some cutaneous human papillomaviruses (HPV). This fact, together with the composition of cis-responsive elements in its long control region and phylogenetic evaluation of segments of its E6, E1, and L1 genes, indicates a relationship of MnPV to the cottontail rabbit papillomavirus and several HPV types found in lesions of cutaneous epithelia, in particular to those that are associated with epidermodysplasia verruciformis. MnPV may be a useful model system for tumorigenesis of cutaneous epithelia in humans.
Neil D Christensen - One of the best experts on this subject based on the ideXlab platform.
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intracutaneous dna vaccination with the e8 gene of cottontail rabbit papillomavirus induces protective immunity against virus challenge in rabbits
Journal of Virology, 2002Co-Authors: Ricai Han, Martin D. Pickel, Nancy M Cladel, Neil D ChristensenAbstract:The cottontail rabbit papillomavirus (CRPV)-rabbit model has been used in several studies for testing prophylactic and therapeutic papillomavirus vaccines. Earlier observations had shown that the CRPV nonstructural genes E1, E2, and E6 induced strong to partial protective immunity against CRPV infection. In this study, we found that CRPV E8 immunization eliminated virus-induced papillomas in EIII/JC inbred rabbits (100%) and provided partial protection (55%) against virus challenge in outbred New Zealand White rabbits. CRPV-E8 is a small open reading frame, coding for a 50-amino-acid protein, that is colinear with the CRPV E6 gene and has features similar to those of the bovine papillomavirus and human papillomavirus E5 genes. Papillomas that grew on E8-vaccinated outbred rabbits were significantly smaller than those on vector-vaccinated rabbits (P < 0.01; t test). Delayed-type hypersensitivity skin tests showed that some of the E8-vaccinated rabbits had positive responses to E8-specific peptides.
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protection of rabbits from viral challenge by gene gun based intracutaneous vaccination with a combination of cottontail rabbit papillomavirus e1 e2 e6 and e7 genes
Journal of Virology, 1999Co-Authors: Ricai Han, Cynthia A Reed, Nancy M Cladel, Xuwen Peng, Neil D ChristensenAbstract:In this study, cottontail rabbit papillomavirus infection of domestic rabbits was used as an animal model to develop papillomavirus early gene-based vaccines. Groups of rabbits were intracutaneously vaccinated with single papillomavirus early genes E1, E2, E6, and E7 or with a combination of these four genes. Only a fraction of rabbits were protected from subsequent viral challenge when vaccinated with the E1 or E6 gene. Viral tumor growth in those rabbits vaccinated with the E1 or E2 gene was suppressed compared to that in controls. In contrast, seven of nine rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge. These data indicated that intracutaneous genetic vaccination with the combination of the E1, E2, E6, and E7 genes can be an effective strategy for immunoprophylaxis of papillomavirus infection.
Ricai Han - One of the best experts on this subject based on the ideXlab platform.
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intracutaneous dna vaccination with the e8 gene of cottontail rabbit papillomavirus induces protective immunity against virus challenge in rabbits
Journal of Virology, 2002Co-Authors: Ricai Han, Martin D. Pickel, Nancy M Cladel, Neil D ChristensenAbstract:The cottontail rabbit papillomavirus (CRPV)-rabbit model has been used in several studies for testing prophylactic and therapeutic papillomavirus vaccines. Earlier observations had shown that the CRPV nonstructural genes E1, E2, and E6 induced strong to partial protective immunity against CRPV infection. In this study, we found that CRPV E8 immunization eliminated virus-induced papillomas in EIII/JC inbred rabbits (100%) and provided partial protection (55%) against virus challenge in outbred New Zealand White rabbits. CRPV-E8 is a small open reading frame, coding for a 50-amino-acid protein, that is colinear with the CRPV E6 gene and has features similar to those of the bovine papillomavirus and human papillomavirus E5 genes. Papillomas that grew on E8-vaccinated outbred rabbits were significantly smaller than those on vector-vaccinated rabbits (P < 0.01; t test). Delayed-type hypersensitivity skin tests showed that some of the E8-vaccinated rabbits had positive responses to E8-specific peptides.
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protection of rabbits from viral challenge by gene gun based intracutaneous vaccination with a combination of cottontail rabbit papillomavirus e1 e2 e6 and e7 genes
Journal of Virology, 1999Co-Authors: Ricai Han, Cynthia A Reed, Nancy M Cladel, Xuwen Peng, Neil D ChristensenAbstract:In this study, cottontail rabbit papillomavirus infection of domestic rabbits was used as an animal model to develop papillomavirus early gene-based vaccines. Groups of rabbits were intracutaneously vaccinated with single papillomavirus early genes E1, E2, E6, and E7 or with a combination of these four genes. Only a fraction of rabbits were protected from subsequent viral challenge when vaccinated with the E1 or E6 gene. Viral tumor growth in those rabbits vaccinated with the E1 or E2 gene was suppressed compared to that in controls. In contrast, seven of nine rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge. These data indicated that intracutaneous genetic vaccination with the combination of the E1, E2, E6, and E7 genes can be an effective strategy for immunoprophylaxis of papillomavirus infection.
P F Lambert - One of the best experts on this subject based on the ideXlab platform.
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Role of transcriptional repressors in transformation by bovine papillomavirus type 1.
Journal of Virology, 1994Co-Authors: T R Zemlo, B Lohrbach, P F LambertAbstract:Abstract Transformation of rodent cells by bovine papillomavirus type 1 (BPV-1) has been shown to require the direct contribution of the viral oncogenes encoded by the E5, E6, and E7 translational open reading frames (ORFs). It is also known that the viral E1 and E2 ORFs contribute indirectly to cellular transformation through their transcriptional modulation of these viral oncogenes. A mutant BPV-1 disrupted in two of the proteins encoded by the E2 ORF, the E2 transcriptional repressors, has a complex transformation phenotype. In this paper, we provide evidence that (i) this phenotype is not attributable to the absence of other viral genes that overlap the E2 repressor genes, (ii) the acquisition of transformation over time in cells harboring the E2 double-repressor mutant correlates with reversions of the mutations that disrupt one of the E2 repressor genes, E8/E2TR, and (iii) the initial transformation defect in the E2 double-repressor mutant can be rescued by disruption of the full-length protein encoded by the E1 ORF, the E1 transcriptional repressor. We propose a model by which the combination of E1 and E2 transcriptional repressors functions to modulate the transforming capacity of the virus.
Nancy M Cladel - One of the best experts on this subject based on the ideXlab platform.
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intracutaneous dna vaccination with the e8 gene of cottontail rabbit papillomavirus induces protective immunity against virus challenge in rabbits
Journal of Virology, 2002Co-Authors: Ricai Han, Martin D. Pickel, Nancy M Cladel, Neil D ChristensenAbstract:The cottontail rabbit papillomavirus (CRPV)-rabbit model has been used in several studies for testing prophylactic and therapeutic papillomavirus vaccines. Earlier observations had shown that the CRPV nonstructural genes E1, E2, and E6 induced strong to partial protective immunity against CRPV infection. In this study, we found that CRPV E8 immunization eliminated virus-induced papillomas in EIII/JC inbred rabbits (100%) and provided partial protection (55%) against virus challenge in outbred New Zealand White rabbits. CRPV-E8 is a small open reading frame, coding for a 50-amino-acid protein, that is colinear with the CRPV E6 gene and has features similar to those of the bovine papillomavirus and human papillomavirus E5 genes. Papillomas that grew on E8-vaccinated outbred rabbits were significantly smaller than those on vector-vaccinated rabbits (P < 0.01; t test). Delayed-type hypersensitivity skin tests showed that some of the E8-vaccinated rabbits had positive responses to E8-specific peptides.
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protection of rabbits from viral challenge by gene gun based intracutaneous vaccination with a combination of cottontail rabbit papillomavirus e1 e2 e6 and e7 genes
Journal of Virology, 1999Co-Authors: Ricai Han, Cynthia A Reed, Nancy M Cladel, Xuwen Peng, Neil D ChristensenAbstract:In this study, cottontail rabbit papillomavirus infection of domestic rabbits was used as an animal model to develop papillomavirus early gene-based vaccines. Groups of rabbits were intracutaneously vaccinated with single papillomavirus early genes E1, E2, E6, and E7 or with a combination of these four genes. Only a fraction of rabbits were protected from subsequent viral challenge when vaccinated with the E1 or E6 gene. Viral tumor growth in those rabbits vaccinated with the E1 or E2 gene was suppressed compared to that in controls. In contrast, seven of nine rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge. These data indicated that intracutaneous genetic vaccination with the combination of the E1, E2, E6, and E7 genes can be an effective strategy for immunoprophylaxis of papillomavirus infection.