Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences.

The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.     

Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation

Papillomaviruses are small DNA viruses that are associated with benign and malignant epithelial lesions, including >95% of cervical cancers and approximately 20% of head and neck cancers. Because papillomavirus replication and virion production are tied to epithelial cell differentiation, infectious papillomavirus virion production has been limited to cumbersome organotypic cultures and mouse xenografts. Consequent difficulties in obtaining useful amounts of wild-type or mutant human papillomavirus (HPV) virions have greatly limited studies on many aspects of papillomavirus biology. To overcome these limitations, we developed a system to encapsidate the full-length papillomaviral genome into infectious virions, independently of viral DNA replication and epithelial differentiation. This transient-transfection-based system produces >1,000 times more infectious virus per cell culture dish than the much more labor-intensive organotypic culture. Furthermore, we show that this method allows the facile generation of infectious particles containing wild-type, mutant, or chimeric papillomaviral genomes, overcoming barriers to studying many facets of replication, host interactions, and vaccine and drug development, which has been limited by the insufficient availability of infectious virions.     

Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism

Human papillomavirus type 16 (HPV16) infection is a major risk factor for the development of squamous cell cancers of the cervix and of the head and neck. A major barrier to understanding the progression from initial infection to cancer has been the lack of in vitro models that allow infection, replication, and persistence of the viral genome as an episome in differentiated epithelial cells. To overcome this barrier, we designed an adenoviral delivery vector that contained a full HPV16 genome flanked by LoxP homologous recombination sites and a fluorescent reporter that was expressed only after the HPV genome was excised by Cre recombinase. This system delivered circular HPV16 genomes to cervical epithelial cells and well differentiated human airway epithelia. After delivery, the HPV16 genome replicated and persisted as an episome in cervical keratinocytes. These cells developed an immortalized phenotype and a dysplastic epithelial appearance. Moreover, induction of differentiation led to the expression of late genes and production of infectious HPV16 virions. This work provides a means of introducing biologically active HPV genomes into epithelial cells, which are normally difficult to transfect. These methods allow the study of HPV genome replication and gene expression in the earliest stages of HPV genome establishment, and they may provide a means to study nononcogenic HPV viral types.     

Efficient transactivation and morphologic transformation by bovine papillomavirus genes expressed from a bovine papillomavirus/simian virus 40 recombinant virus.

To efficiently introduce bovine papillomavirus type 1 genes into cultured cells, we constructed a hybrid viral genome in which the simian virus 40 early region is replaced with a segment of the bovine papillomavirus type 1 transforming region. High-titer stocks of simian virus 40 virions containing the recombinant genome were produced in monkey cells that express simian virus 40 large tumor antigen. Cells infected with this virus efficiently expressed the bovine papillomavirus type 1 E2 and E5 genes. Expression of the E2 gene caused transactivation of genes linked to the bovine papillomavirus type 1 control region, resulting in up to a 1000-fold induction. At high multiplicity of infection of a cell line containing an integrated reporter gene, most cells were infected and responded to transactivation. Within 48 hr of infection with wild-type virus but not with an open reading frame E5 mutant, mouse C127 cells displayed dramatic changes in morphology and growth characteristics similar to those seen in tumorigenic transformation. This system can be used to determine the acute cellular response to introduction of bovine papillomavirus type 1 transforming and regulatory genes; it can also be used to induce foreign genes stably incorporated into cultured mammalian cells.     

DNA bending is induced in an enhancer by the DNA-binding domain of the bovine papillomavirus E2 protein.

The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I "footprinting" on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucleotide) as the E2 binding site. Using electrophoretic methods we have shown that the DNA-binding domain changes conformation of the enhancer by inducing significant DNA bending.     

Characterization of integrated hepatitis B viral DNA cloned from a human hepatoma and the hepatoma-derived cell line PLC/PRF/5.

Recombinant phage clones carrying integrated hepatitis B virus (HBV) DNA sequences have been isolated from two phage libraries made from human DNA of a hepatoma and a hepatoma-derived cell line. One clone from each library has been characterized both by restriction mapping and by electron microscopy. In one clone there is at least one complete and uninterrupted HBV genome, and in the other the HBV sequences are composed of two major subgenomic fragments inverted with respect to each other. The host-virus junctions are localized within the positions 1,700-2,600 base pairs on the physical map of the free viral genome. The pre-S/S (surface antigen gene) region is conserved between the two clones. The two clones do not have common cellular sequences nor do they contain cellular homologues to six retroviral oncogenes. For one clone, the hepatitis B surface antigen gene was found to be functional when introduced into mouse thymidine kinase-negative cells by transfection.     

Excretion of hepatitis B surface antigen particles from mouse cells transformed with cloned viral DNA.

A plasmid containing two cloned hepatitis B virus genomes in a tandem head-to-tail arrangement has been introduced into mouse fibroblasts by using cotransformation with the cloned herpes simplex virus thymidine kinase gene. Several copies of the plasmid were integrated into high molecular weight cellular DNA. The original tandem structure of the hepatitis B virus DNA was conserved. Hepatitis B surface antigen was synthesized by all the 15 clones examined. The other viral antigens were not detected. The surface antigen was excreted into the cell culture medium as particles having the same characteristics as those found in human serum. It is estimated that 2-4 X 10(4) particles were produced per mouse cell per 24 hr in two clones. This value corresponds to approximately 2-4 X 10(6) surface antigen polypeptides per cell per 24 hr.     

Detection of Anaplasma-marginale-infected tick vectors by using a cloned DNA probe.

Anaplasmosis is the most widely distributed of several important tick-borne diseases that constrain cattle production throughout much of the world. Evaluation of the effectiveness of disease control strategies that integrate vaccination with tick control requires the ability to monitor tick and cattle infection rates. To detect Anaplasma marginale in ticks and bovine erythrocytes, a 2-kilobase DNA fragment from a cloned A. marginale gene coding for a surface protein having a Mr of 105,000 was prepared and evaluated as a probe. The probe was species specific and detected A. marginale DNA derived from infected bovine erythrocytes and adult Dermacentor ticks infected either as nymphs or adults. Tick infection was confirmed by microscopy and test feeding on a susceptible calf. The sensitivity of the probe is suitable for detecting infected ticks in experimental and field epizootiology studies.     

Differentiation of schistosomes by species, strain, and sex by using cloned DNA markers.

We have detected species, strain, and sex-specific genetic markers for the genus Schistosoma by Southern blot analysis of its DNA using cloned DNA segments of the Schistosoma mansoni ribosomal gene as probes. Restriction analysis of DNA from eight different strains of S. mansoni, from Africa and the Caribbean, revealed that the predominant or major DNA fragment containing the ribosomal gene unit was the same in each but that low copy number or minor fragments containing the gene varied. It was shown that the detection of these minor fragments could serve as the basis for both strain differentiation and the analysis of individual differences within a strain. Analysis of the parents and progeny of a genetic cross revealed sex-linked markers and suggested that these markers are inherited in a Mendelian fashion. DNAs from the species Schistosoma haematobium and Schistosoma japonicum were also analyzed. Differences in the length of the major repeating unit of the ribosomal gene served to distinguish each species. Furthermore, an array of minor bands was detected in each species, suggesting that strains of S. haematobium and S. japonicum could be differentiated in the same manner as S. mansoni strains.     

Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase gamma.     

Expression of a functional influenza viral cap-recognizing protein by using a bovine papilloma virus vector.

The gene for the influenza viral PB2 protein, which recognizes and binds the 5'-terminal cap 1 structures (m7GpppNm) on eukaryotic mRNAs, was inserted into a bovine papilloma virus vector under the control of a mouse metallothionein I (MT-I) promoter. After transfection of this vector into mouse NIH 3T3 cells, a cell line, cPB2, was obtained that produces PB2-specific mRNA and authentic PB2 protein. Induction of the MT-I promoter with CdCl2 causes about a 10-fold increase in PB2 mRNA and protein levels. The expressed PB2 protein is functional, as it relieves the block in viral mRNA synthesis exhibited by a temperature-sensitive viral mutant containing a cap-binding defect in the PB2 protein. This demonstrates complementation of a function of a negative-strand RNA virus by a gene product expressed in a cell line from recombinant DNA.     

Induction of structural changes in the bovine papillomavirus type 1 origin of replication by the viral E1 and E2 proteins.

Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 E1 and E2 proteins with the viral origin of replication (ori). E1 was found to generate significant distortions to the structure of ori, as assayed by KMnO4 oxidation of DNA. The primary site of ori distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of E1 required for ori distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for E1 binding to ori, as assayed by nuclease protection. Under these conditions, E1 bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of E1 were required for binding to the adjacent ori AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of E1 to ori.     

Analysis of beta-thalassemia mutations in northern Thailand using an automated fluorescence DNA sequencing technique

A total of 218 beta-thalassemia (thal) genes from 109 beta-thal major patients were characterized using an automated fluorescence DNA sequencing technique. Eight different mutations were identified in all 218 alleles (100%). Four common mutations accounted for 96.8% [49.5% were codons 41/42 (-TTCT), 34.4% were codon 17 (A --> T), 6.9% were IVS-I-1 (G --> T) and, 6.0% were codons 71/72 (+A)]. There were three cases of -28 (A --> G) and one of IVS-II-654 (C --> T), mutations that have been previously described in Thai subjects. We also identified two mutations in the beta-globin promoter region which have not been reported in Thailand before [-31 (A --> G) and -87 (C --> A)]. Although these mutations are described as beta+-thal, the compound heterozygote with one of the common beta(o)-thal mutations exhibits the phenotype of beta-thal major. The frequency of beta-thal genes in northern Thailand were similar to the northeastern region, but different from those reported in southern and central Thailand, where IVS-I-5 (G --> C) and IVS-II-654 (C --> T) were the second most common anomalies, respectively. The spectrum of beta-globin gene mutations from this study will be useful for planning a prenatal diagnosis program especially for this region of Thailand.     

Detection of human papillomavirus in cervical swabs from Indian women by cytological and immunocytochemical technique.

Cervical swabs were collected from 88 women in the age group of 17-55 years in Calcutta, India to study the prevalence of human papillomavirus (HPV) infection. The viral infection was demonstrated on the basis of koilocytotic changes in Papanicolaou staining (PAP test) and the presence of viral capsid antigen as detected by immunocytochemistry (IMC test). The PAP and IMC tests revealed 25% and 28.4% of women, respectively, to be positive for HPV infection. Higher frequency (61.1% to 87.5%) of infection was observed in moderate to severe dysplastic lesions. HPV infection was observed to be more frequent in the younger age group (less than 40 years) with 1-2 parity. Although koilocytes were absent in all (n = 51) of the apparently normal subjects, 4 (7.8%) of them were positive for HPV antigen. Except for these, sensitivity of PAP test and IMC test for the detection of HPV was found to be almost similar.     

Isolation of a human papillomavirus from a patient with epidermodysplasia verruciformis: presence of related viral DNA genomes in human urogenital tumors.

The DNA genome of a human papillomavirus (HPV), tentatively designated HPV-EV, was molecularly cloned from hand to leg lesions of a patient with epidermodysplasia verruciformis, a chronic skin disease associated with a 30% risk of developing cancer. Using stringent hybridization conditions, we observed less than 5% homology between HPV-EV and the cloned genomes of HPV-1, HPV-4, HPV-5, and HPV-5a. HPV-EV DNA showed approximately 6% homology with HPV-2 and 36% homology with HPV-3. These data suggest that HPV-EV is partially related to HPV-3. Using 32P-labeled cloned HPV-EV as probe in Southern blot hybridization experiments, we detected HPV-EV-related DNA in the carcinoma in situ (Bowenoid lesion) of the vulva of the patient from which HPV-EV was isolated. HPV-EV-related DNA was detected in 2 of 10 vulva carcinomas and in 2 of 31 cervical carcinomas. Related DNA sequences were found in papillomas from each of two patients with condyloma acuminata (anogenital warts), which is of interest considering that condylomas have been reported to convert occasionally to carcinomas. The positive vulva DNAs were also probed with other cloned HPV DNAs: HPV-1, HPV-4, and HPV-5a-related sequences were not detected; HPV-3 and HPV-2 DNA probes detected strong and weak DNA bands, respectively, of the same size as found with HPV-EV. The HPV DNA sequences were present in the positive tumors mainly as free viral DNA molecules; no evidence for integration into cellular DNA was found. The emerging biological picture with papillomaviruses is that cells transformed by these viruses are maintained in a transformed state by free episomal genomes. Thus, our findings are consistent with the idea, but by no means establish, that HPVs play a role in human cancer by a similar mechanism.     

Measurement of hepatitis B viral DNA in serum by solution hybridization and comparison with the dot-blot hybridization technique

A recently introduced method for the detection of hepatitis B viral (HBV) DNA in the serum, using solution hybridization, was assessed for its sensitivity and specificity in 242 sera from patients with chronic HBV infection, and in controls. In 87 sera the results were compared with the classical dot-blot technique. The new method gave positive results in 82% (56/68) of all dot-blot-positive sera and in 97% (35/36) of those with strong positive dot-blot reactions. All 30 hepatitis B surface antigen (HBsAg) carriers positive for hepatitis B e antigen (HBeAg) were positive in the solution hybridization assay, with a mean HBV-DNA level of 172 +/- SE 36 pg/ml. Negative results were obtained in all 20 subjects negative for all HBV markers and in all 36 HBsAg+/anti-HBe+ carriers with normal aminotransferase activity, negative for hepatitis B core antigen (HBcAg) expression in the liver. By contrast 93% (66/71) of the anti-HBe+ patients with positive HBcAg expression in the liver were positive for serum HBV-DNA in the solution hybridization assay, with a mean level of 30.5 +/- SE 6.7 pg/ml. Of 14 sera with discrepant HBV-DNA results in the two assays, 12 were solution hybridization-negative/dot-blot-positive and 2 were solution hybridization-positive/dot-blot-negative. These results indicate that the new, simple method is specific, and suitable for the determination of serum HBV-DNA in clinical practice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpesvirus-dependent amplification and inversion of cell-associated viral thymidine kinase gene flanked by viral a sequences and linked to an origin of viral DNA replication.

The genome of herpes simplex virus 1 or 2 consists of two components, L and S, which invert relative to each other during infection. As a result, viral DNA consists of four equimolar populations of molecules differing solely in the relative orientations of the L and S components. Previous studies have shown that the a sequences, located in the same orientation at the genomic termini and in inverted orientation at the L-S junction, play a key role in the inversion of L and S components. In this report we describe a virus-dependent system designed to allow identification of the viral genes capable of acting in trans to invert DNA flanked by inverted copies of a sequences. In this system, cells are converted to the thymidine kinase-positive phenotype with a chimeric plasmid carrying the thymidine kinase gene flanked by inverted copies of the a sequence and linked to an origin of viral DNA replication derived from the S component. The DNA introduced into the cells is retained and propagated in its original sequence arrangement as head-to-tail concatemers. Infection of these cells with herpes simplex virus 1 or 2 results in as much as 100-fold amplification of the plasmid sequences and inversion of the DNA flanked by copies of the a sequence. In infected cells, the amplified resident DNA accumulates in head-to-tail concatemers and no rearrangement other than the inversions could be detected. These results suggest that the a sequence-dependent inversions required trans-acting viral gene products.