Conserved polynucleotide sequences among the genomes of papillomaviruses.

The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.     

Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.     

Isothermal strand-displacement amplification applications for high-throughput genomics

Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.     

Characterization of two novel polyomaviruses of birds by using multiply primed rolling-circle amplification of their genomes

Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds--avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)--are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5' region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses.     

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.     

Cloning and characterization of a papillomavirus associated with papillomas and carcinomas in the European harvest mouse (Micromys minutus).

Individuals in a colony of European harvest mice (Micromys minutus) were diagnosed with a variety of skin tumors including papillomas, trichoepitheliomas, and sebaceous carcinomas. Papillomavirus group-specific antigens and viruslike particles were detected in the papillomas. A 7.6-kilobase supercoiled circular DNA, which was cleaved once by EcoRI, was visualized in papilloma extracts by low-stringency Southern blot hybridization with a bovine papillomavirus type 2 probe. The molecule was cloned in pUC18, and a restriction map was generated. The molecule was shown to be colinear with the genome of human papillomavirus type 1a by partial sequence analysis. The DNA hybridized to human papillomavirus type 1a, rabbit oral papillomavirus, and the genome of Mastomys natalensis papillomavirus at Tm - 33 degrees C but not to the DNAs of 13 other papillomaviruses. Transformation of NIH 3T3 or C127I cells by tail papilloma extracts or transfected viral DNA was not observed. All 17 tumors examined contained large amounts of viral DNA in a supercoiled, unintegrated form as revealed by Southern blot hybridization. Furthermore, many extracts (25 of 35) from normal organs and skin of individuals with lesions elsewhere on their bodies contained viral DNA. This represents the first reported molecular cloning of a papillomavirus genome from a mouse species.     

Human papillomaviruses associated with epidermodysplasia verruciformis. II. Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes.

The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to types 3 and 5, respectively. These viruses are shown in the present study to be different from all of the HPV types so far characterized; they have tentatively been named HPV-10 and HPV-12. The HPV-3a, HPV-8, and HPV-12 DNAs and the two SalI fragments of HPV-10 DNA (94.1 and 5.9% of the genome length) were cloned in Escherichia coli after having been inserted in plasmid pBR322. The cloned HPV genomes have similar sizes (about 7,700 base pairs), but their guanine-plus-cytosine contents differ from 41.8% for HPV-12 DNA to 45.5% for HPV-3a DNA. The study of the sensitivity of the four HPV DNAs to 14 restriction endonucleases permitted the construction of cleavage maps. Evidence for conserved restriction sites was found only for the HPV-3a and HPV-10 genomes since 5 of the 21 restriction sites localized in the HPV-3a DNA seem to be present also in the HPV-10 DNA. Hybridization experiments, performed in liquid phase at saturation, showed a 35% sequence homology between HPV-3a and HPV-10 DNAs, 17 to 29% sequence homology among HPV-5, HPV-8, and HPV-12 DNAs, almost no sequence homology between the HPV-3a or HPV-10 DNA and the other HPV DNAs, and a weak homology between HPV-9 DNA and HPV-8 or HPV-12 DNA. Blot hybridization experiments showed no sequence homology between the HPV-3a, HPV-8, and HPV-12 DNAs and the DNAs of the HPVs associated with skin warts (HPV-1a, HPV-2, HPV-4, and HPV-7) or with mucocutaneous and mucous membrane lesions (HPV-6b and HPV-11a, respectively). One exception was a weak sequence homology between the HPV-2 prototype and HPV-3a or HPV-10 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phylogenetic analysis of 48 papillomavirus types and 28 subtypes and variants: a showcase for the molecular evolution of DNA viruses.

Papillomaviruses are attractive models for studying the molecular evolution of DNA viruses because of the large number of isolates that exhibit genomic diversity and host species and tissue specificity. To examine their relationship, we selected two amino acid sequences, one of 52 residues within the early gene E1 and the other of 44 residues within the late gene L1, which allowed insertion- and deletion-free alignment of all accessible papillomavirus sequences. We constructed phylogenetic trees from the amino acid and corresponding nucleotide sequences from 28 published and 20 newly determined animal and human papillomavirus (HPV) genomic sequences by using distance matrix, maximum-likelihood, and parsimony methods. The trees agreed in all important topological aspects. One major branch with two clearly separated clusters contained 11 HPV types associated with epidermodysplasia verruciformis. A second major branch had all the papillomaviruses involved in genital neoplasia and, in distant relationship, the cutaneous papillomaviruses HPV type 2a (HPV-2a), HPV-3, and HPV-10 as well as the "butcher's" papillomavirus HPV-7 and two simian papillomaviruses. Four artiodactyl (even-toed hoofed mammal) papillomaviruses, the cottontail rabbit papillomavirus, and avian (chaffinch) papillomavirus type 1 formed a third major branch. Last, four papillomaviruses exhibited little affinity to any of these three branches; these were the cutaneous types HPV-1a, HPV-4, and HPV-41 and B-group bovine papillomavirus type 4. The phylogeny suggests that some branches of papillomavirus evolution are restricted to particular target tissues and that a general process of long-term papillomavirus-host coevolution has occurred. This latter hypothesis is still conjectural because of bias in the current data base for human types and the paucity of animal papillomavirus sequences. The comparison of evolutionary distances for the most closely related types with those of 28 subtypes and variants of HPV-2, HPV-5, HPV-6, HPV-16, and HPV-18 supports the type as a natural taxonomic unit, with subtypes and variants being expressions of minor intratype genomic diversity similar to that found in the natural populations of all biological species. An exception to this seems to be HPV-2c, which has an evolutionary distance from HPV-2a of the intertype magnitude and may eventually have to be regarded as a distinct type. We describe an experimental approach that estimates the taxonomic and phylogenetic positions of newly identified papillomaviruses without viral isolation and complete genomic sequencing.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partitioning Viral Genomes in Mitosis: Same Idea,Different Targets

Papillomavirus infections are long-lived and persistent. The circular DNA of the viral genome is maintained in dividing epithelial cells as an extrachromosomal element. The E2 protein of the virus binds to the viral genome and tethers it to mitotic chromosomes to ensure that the genome is retained and faithfully partitioned in dividing cells. This mechanism has been best studied for bovine papillomavirus type 1. Recent evidence indicates that while this is a common strategy among papillomaviruses, different viruses have evolved different chromosomal targets.     

Transient replication of human papillomavirus DNAs.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.     

Molecular cloning and characterization of the DNAs of human papillomaviruses 19, 20, and 25 from a patient with epidermodysplasia verruciformis.

Five human papillomavirus (HPV) DNAs from lesions of an epidermodysplasia verruciformis patient were cloned in lambda L 47: DNA of HPV 5, which predominated in the carcinoma; DNA of a variant type of HPV 8, which was not detected in the carcinoma DNA by Southern blot hybridization but only by cloning; and DNAs of three papillomaviruses that were isolated from warts. Southern blot and liquid phase DNA-DNA hybridization under stringent conditions showed that the three viruses from warts were new types, which we named HPVs 19, 20, and 25. These viruses cross-hybridized between 3 and 29% among themselves and with HPVs 5 and 8. After physical mapping with several restriction enzymes, the colinear genomes were aligned with HPV 8 DNA to define early and late regions. HPVs 8, 19, and 25 shared homology in different parts of their genomes.     

A novel virus detected in papillomas and carcinomas of the endangered western barred bandicoot (Perameles bougainville) exhibits genomic features of both the Papillomaviridae and Polyomaviridae

Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size ( approximately 7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families.     

Analysis of a restriction endonuclease map for a rabbit papillomavirus DNA

A restriction endonuclease map was constructed for rabbit papillomavirus DNA. Analysis of the viral genome by cleavage with specific endonucleases, when compared with bovine papillomavirus types 1 and 2 genome maps, revealed similarities among the three genomes. A number of cleavage sites and their relative order on each genome were found to be conserved.     

Biochemical characterization of two types of human papillomaviruses associated with epidermodysplasia verruciformis.

The DNAs of the human papillomaviruses (HPVs) associated with the benign lesions of two patients suffering from epidermodysplasia verruciformis (patients JD and JK) were analyzed by using 12 restriction endonucleases. None of the restriction endonucleases were one-cut enzymes for the HPV DNA obtained from patient JD, referred to as the prototypical HPV-5, whereas five of them were one-cut enzymes for the DNA of the major virus found in patient JK, referred to as HPV-9. The molecular cloning of the two fragments resulting from the cleavage of HPV-5 DNA by endonuclease HindIII and of the single fragment obtained after treatment of HPV-9 DNA with endonuclease BamHI was performed in Escherichia coli after the fragments were inserted in plasmid pBR322. A cleavage map of the two cloned genomes was constructed. Little sequence homology (4 to 5%) was detected between HPV-5 and HPV-9 DNAs by DNA-DNA hybridization experiments in liquid phase at saturation; this homology was reproducibly higher than that (2 to 3%) detected under the same conditions between these DNAs and HPV-1a DNA. In addition, blot hybridization experiments performed under stringent conditions showed no or little sequence homology between the DNAs of HPV-5 and HPV-9 and those of HPV prototypes of types 1, 2, 3, 4, and 7 associated with skin warts. These results confirm that HPV-5 and HPV-9 are two distinct HPV types.