Isolation and cloning of the raccoon (Procyon lotor) papillomavirus type 1 by using degenerate papillomavirus-specific primers
Annabel RectorKoenraad Van DoorslaerMads F. BertelsenIan K. BarkerRolf-Arne ØlbergPhilippe LemeyJohn P. SundbergMarc Van Ranst
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Partial sequences of a novel papillomavirus were amplified from a cutaneous lesion biopsy of a raccoon ( Procyon lotor ), by using PCR with degenerate papillomavirus-specific primers. The Procyon lotor papillomavirus type 1 (PlPV-1) DNA was amplified with long template PCR in two overlapping fragments, together encompassing the entire genome, and the complete PlPV-1 genomic sequence was determined. The PlPV-1 genome consists of 8170 bp, and contains the typical papillomaviral open reading frames, encoding five early proteins and two late capsid proteins. Besides the classical non-coding region (NCR1) between the end of L1 and the start of E6, PlPV-1 contains an additional non-coding region (NCR2) of 1065 bp between the early and late protein region, which has previously also been described for the canine oral papillomavirus (COPV) and the Felis domesticus papillomavirus (FdPV-1). Phylogenetic analysis places PlPV-1 together with COPV and FdPV-1 in a monophyletic branch which encompasses the Lambda papillomavirus genus.Keywords:
Bovine papillomavirus
Coding region
Papillomaviridae
Bovine papillomavirus
Southern blot
Homology
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Papillomaviridae
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Human papillomavirus (HPV) capsids are formed through a network of inter- and intra-pentameric hydrophobic interactions and disulfide bonds. 72 pentamers of the major capsid protein, L1, and an unknown amount of the minor capsid protein, L2, form the structure of the capsid. There are 12 conserved L1 cysteine residues in HPV16. While C175, C185, and C428 have been implicated in the formation of a critical inter-pentameric disulfide bond, no structural or functional roles have been firmly attributed to any of the other conserved cysteine residues. Here, we show that substitution of cysteine residues C161, C229, and C379 for serine hinders the accumulation of endonuclease-resistant genomes as virions mature within stratifying and differentiating human epithelial tissue. C229S mutant virions form, but are non-infectious. These studies add detail to the differentiation-dependent assembly and maturation that occur during the HPV16 life cycle in human tissue.
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A series of mutations In open reading frame (ORF) E5 of bovine papillomavirus type 1 has been constructed to determine whether this putative gene is required for in vitro oncogenic transformation by viral DNA. Frameshift mutations at either of two different positions located exclusively in ORF E5 cause a substantial reduction In the ability of the cloned viral DNA to induce the appearance of transformed foci of mouse C127 cells. A genetic mapping experiment with one of the mutants indicates that this characteristic transformation defect is actually due to the constructed mutation in ORF E5. Analysis of 10 different mutants with sequence changes at a single position in the ORF showed that there is an exact correspon- dence between transformation-competence and the ability of the 3' half of ORF E5 to be correctly translated. The trans- formation defect of an ORF E5 frameshift mutant can be suppressed by a second mutation that restores the correct reading frame to most of the ORF, but not by one that restores the reading frame near the 3' end of the ORF. These results constitute strong genetic evidence that translation of ORF E5 is required for efficient transformation of mouse C127 cells by bovine papillomavirus DNA. Wild-type ORF E5 has the potential to encode a short hydrophobic protein or polypeptide domain.
Bovine papillomavirus
Reading frame
ORFS
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Coding region
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SUMMARY A coding sequence at the 5′ end of mRNA 4 of the coronavirus MHV-JHM was determined by M13/chain-terminator sequencing of cloned cDNA. An open reading frame of 417 bases with the potential to encode a polypeptide of mol. wt. 15200 (139 residues) was identified. The 3′ end of the open reading frame overlapped by 16 bases the start of an open reading frame found in mRNA 5. The translation product of mRNA 4 was predicted to be a basic polypeptide rich in threonine. It had a large hydrophobic region near the amino terminus and a basic carboxy terminus. An intracellular, virus-specific polypeptide, which has been previously described as having a mol. wt. of 14000 to 14500 has the size and charge characteristics of such a translation product.
Coronavirus
Coding region
2019-20 coronavirus outbreak
Sequence (biology)
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A series of mutations in open reading frame (ORF) E5 of bovine papillomavirus type 1 has been constructed to determine whether this putative gene is required for in vitro oncogenic transformation by viral DNA. Frameshift mutations at either of two different positions located exclusively in ORF E5 cause a substantial reduction in the ability of the cloned viral DNA to induce the appearance of transformed foci of mouse C127 cells. A genetic mapping experiment with one of the mutants indicates that this characteristic transformation defect is actually due to the constructed mutation in ORF E5. Analysis of 10 different mutants with sequence changes at a single position in the ORF showed that there is an exact correspondence between transformation-competence and the ability of the 3' half of ORF E5 to be correctly translated. The transformation defect of an ORF E5 frameshift mutant can be suppressed by a second mutation that restores the correct reading frame to most of the ORF, but not by one that restores the reading frame near the 3' end of the ORF. These results constitute strong genetic evidence that translation of ORF E5 is required for efficient transformation of mouse C127 cells by bovine papillomavirus DNA. Wild-type ORF E5 has the potential to encode a short hydrophobic protein or polypeptide domain.
Bovine papillomavirus
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ORFS
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Bovine papillomavirus type 1 (BPV-1) readily transforms mouse C127 cells, conferring the ability to grow in soft agar and to form tumors in athymic (nu/nu) mice. Electrophoresis of total cellular proteins from these BPV-transformed lines on ultra-high resolution, giant two-dimensional gels displays the presence of novel, papillomavirus-related protein phenotypes. Analysis of the established BPV-1-transformed C127 cell lines, ID13 and ID14, reveals a set of six proteins which are either absent or synthesized at extremely low levels in the parental cell line. One of these proteins is also present in v-ras-transformed C127 cells, but none of the others are found in cells transformed by a variety of viral oncogenes, including the polyomavirus middle T, v-mos, or v-fes. The genome of BPV-1 contains two separate open reading frames (ORFs), E5 and E6, which can act independently to transform C127 cells. In addition, trans-activator and repressor proteins encoded respectively by the full-length and carboxy-terminal E2 ORF regulate the level of expression of other BPV-1 genes. We examined 34 cell lines transformed by intact and subgenomic recombinant DNAs of BPV-1. Cells harboring BPV-1 DNAs engineered to eliminate the expression of ORFs E4, E5, E6, or E7 display five of the PV-associated proteins, but these proteins are not seen in lines lacking the full E2 ORF. Moreover, G418-selected nontransformed cells expressing E2 cDNA from an SV40 promoter exhibit these proteins at high levels. Surprisingly, these proteins are also present in cells containing BPV-1 DNAs with amino-terminal E2 deletions, suggesting that these PV-associated proteins represent novel cellular responses to a factor encoded within the E2-C gene region.
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Bovine papillomavirus
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Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.
Bovine papillomavirus
Extrachromosomal DNA
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Abstract Analysis of the corrected DNA sequence for the bovine papillomavirus type 4 (BPV4) genome revealed that there is no open reading frame (ORF) that might encode an E6 protein. The other two B subgroup bovine papillomaviruses, BPV3 and BPV6, were found to have the same arrangement of ORFs in this region as BPV4. Thus, we conclude that E6 functions are either not required by these viruses or are performed by another viral (or host) protein. Furthermore, the position that might be expected to be occupied by E6, between the long control region and the E7 ORF, contains the E8 ORF, which has the potential to encode a 42‐residue polypeptide with considerable similarity to the E5 transforming protein of BPV1. Therefore, it appears that during the evolution of the B subgroup of BPVs, genomic rearrangements may have occurred resulting in the present layout of the early ORFs.
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The E2 open reading frame of bovine papillomavirus type 1 has been shown genetically to encode at least three transcriptional regulatory factors, and three E2 specific proteins have been recently identified in virally transformed rodent cells. In this study, the genes encoding these E2 specific proteins have been determined. The 48-kilodalton (kDa) protein was identified as the product of a full-length E2 open reading frame cDNA, which confirmed that this polypeptide is the E2 transactivator. The 31-kDa E2 protein species, which is the most abundant E2 specific polypeptide, was identified by analysis of both bovine papillomavirus type 1 mutants and cDNAs to be the previously identified E2 transcriptional repressor, E2-TR, which results from translation initiation at an internal E2 ATG codon. The smallest E2 protein species, the 28-kDa polypeptide, was identified as the product of the E8/E2 fusion gene which results from translation of a spliced mRNA species.
Bovine papillomavirus
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