Integration and excision of SV40 DNA from the chromosome of a transformed cell

The single insertion of SV40 DNA present in the genome of the 14B line of transformed rat cells has been cloned in procaryotic vectors. Analysis of the clones reveals a complex arrangement of viral sequences in which a small tract of DNA is inverted with respect to the major insertion. The nucleotide sequences at the two junctions show sharp transitions between cellular and viral sequences. The sequences which flank the viral insertion have been used as probes to clone the corresponding genomic sequences from the DNA of untransformed rat cells. Analysis of the structure of these clones shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration. When 14B cells are fused with uninfected simian cells a heterogeneous set of low molecular weight superhelical DNAs containing viral sequences is generated. These have been cloned in procaryotic vectors and their structures have been analyzed. All of them span the origin of SV40 DNA replication and are colinear with various segments of the integrated viral DNA and its flanking sequences. The shorter molecules contain part of the integrated viral genome and cellular sequences from one side of the insertion. They were therefore generated by recombination between the viral DNA and its flanking cellular sequences. The longer molecules contain cellular sequences from both sides of the insertion as well as an entire copy of the integrated viral DNA. They were therefore generated by recombination between the flanking cellular sequences. These results argue strongly against the involvement of specific excision enzymes, and rather are discussed in terms of a model involving replication of the integrated viral DNA followed by recombination for release of integrated viral sequences.     

Microdeletion associated with the integration process of hepatitis B virus DNA

Hepatitis B virus (HBV) DNA is often found in integrated form in hepatocellular carcinomas (HCC) and in non-cancerous liver cells of chronic carriers of HBV. However, the process of integration has not been well understood. Analyses of integrant DNA was expected to give clues. However, the majority of the integrants are products of multistep rearrangements following integrations, and analysis of randomly selected samples do not give clues for understanding the process of primary integrant formation. Therefore, one must select an appropriate integrant(s) that has a simple structure. We surveyed a collection of integrants prepared from many HCC's, and found one integrant that has the simplest structure so far studied: The viral genome is almost complete, is joined to cellular DNA using the cohesive end of the viral DNA, and furthermore, the "left" and "right" flanking cellular DNA's are almost contiguous. Analysis of the unoccupied sites in cellular DNA showed that, although almost contiguous, it has generated a microdeletion (15 base pairs) in the target sequence. This target sequence has a short region of homology to the sequence in the viral genome located close to the junction. One integrant with strikingly similar features has been reported independently. Two similar, but not identical cases from literatures could be added to this category. Therefore, the integrants with these properties may represent a unique category among those prepared from hepatocellular carcinomas. Based on these findings, we propose that this integrant represents the primary product of integration, and discuss the intermediate acting in the process of integration.     

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.     

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from a chronically infected liver

We have isolated and determined the structure of a recombinant clone in lambda phage Charon 30 which contains woodchuck hepatitis virus sequences integrated in woodchuck genomic DNA sequences. This clone, in contrast to previously reported clones (Ogston et al., Cell 29:385-394, 1982), was isolated from a chronically infected liver which never developed hepatocellular carcinoma. Southern blot analysis of viral sequences in the clone in conjunction with electron microscope heteroduplex analysis showed that the integrated viral sequences did not contain internal rearrangements, as have those from hepatomas, but were colinear with the cloned viral genome except for the deletion of approximately 500 base pairs of viral sequences (between positions 1,000 and 1,550 on the viral map). Therefore, the integration was probably a defective genome incapable of supporting viral replication. However, the complete open reading frames coding for the viral X, core, presurface , and surface antigen genes were present, indicating that the viral sequences could code for viral antigens. Southern blot analysis of the normal cellular flanking sequences, using flanking sequence probes from the clone, showed that no detectable rearrangements of cellular DNA (less than 50 base pairs) had occurred at the site of viral integration.     

Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration.

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Integrated polyoma genomes in inducible permissive transformed cells.

Using the approach described by Botchan, Topp, and Sambrook (Cell 9:269-287, 1976), we analyzed the organization of the integrated viral sequences in five clonal isolates from the same permissive, inducible cell line (Cyp line) transformed by the tsP155 mutant of polyoma virus. In all five clones, viral sequences were found that could be assigned to a common integration site, as they were joined to the cellular DNA in the same fashion in every instance. However, the sequences comprised between these points differed markedly from clone to clone, as if cell propagation had been accompanied by amplification or recombination or both within the viral insertion. When the clones were compared, no correlation could be found between the abundance, or the organization, of the integrated viral sequences and the amount, or the nature, of the free viral DNA molecules produced during induction. Altogether, our findings suggest that specific events, occurring during either the excision or the subsequent replication of the integrated viral sequences, are responsible for the predominant production of nondefective viral DNA molecules by permissive transformed cells, such as Cyp cells.     

The genetic organization of integrated hepatitis B virus DNA in the human hepatoma cell line PLC/PRF/5.

Hepatitis B virus (HBV) DNA is often found integrated in the genome of infected human liver cells and is supposed to be related to the development of primary liver carcinoma (PLC). Four clones of HBV DNA-containing sequences derived from DNA of the human PLC-derived cell line PLC/PRF/5 are discussed. The viral sequences show no intricate rearrangements excepting for a duplication and an inversion in one case, and a deletion in another. In all cases integration of the viral DNA was seen to be in a region which is single-stranded in the unintegrated HBV DNA. Sequence homologies between human and viral DNA flanking the integration sites have been detected. That may have a functional role in integration. Nucleotide sequence analyses of regions encompassing the viral-human junctions reveal open reading frames which consist of viral and/or human information. The possible expression of chimeric or cellular proteins may play a role in tumour development, and offers directions for further investigations.     

Infectious and noninfectious recombinant clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA

Ten clones of Charon 4A containing proviruses of spleen necrosis virus, an avian retrovirus, and flanking chicken DNA sequences were isolated and characterized. Some clones gave rise to progeny with viral DNA sequences deleted or duplicated, probably as a result of crossing-over in the 600 bp terminal redundancy in viral DNA. The cellular sequences are different in each clone, indicating that all the proviruses are integrated in different sites in cellular DNA. Six clones are infectious and four are not. All the infectious molecules containing a provirus are of a similar size and are smaller than the noninfectious molecules containing a provirus. The viral DNA is not apparently different in eight clones, but two clones, one infectious and one noninfectious, lack two restriction sites each. Large changes in proviral DNA therefore do not seem responsible for the lack of infectivity of some clones. These results are consistent with the hypothesis that neighboring cellular DNA sequences control proviral expression (infectivity).     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

Chromosomal sites for hepatitis B virus integration in human hepatocellular carcinoma.

The discovery that hepatitis B virus (HBV) integrates into host chromosomes raises the question of whether such viral DNA integration correlates directly with the activation of specific oncogenes or the inactivation of anti-oncogenes. To obtain insight into this problem, we randomly collected HBV integrant samples from different human hepatocellular carcinomas and identified the site of chromosomal integration by using in situ hybridization and/or linkage analysis with the flanking cellular DNAs as probes. Our findings did not specifically identify particular HBV DNA integration sites in chromosomes, although chromosomes 11 and 17 seemed to have more than the average number of integrants.     

Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA.

DNA of hepatitis B virus is found to be integrated into the genome of infected human liver cells and may be related to the development of primary liver carcinoma. We have previously reported the cloning of cellular DNA with integrated HBV sequences from the PLC/PRF/5 cell line which derives from a human primary liver carcinoma. Two clones, designated as A-10.7 and A-10.5, and a third uncloned fragment are compared by restriction enzyme mapping, hybridization and nucleotide sequencing. The results indicate that amplification of integrated viral DNA and host flanking regions has occurred, followed by transposition and/or major deletions. The implications of these findings for the development of primary liver carcinoma are discussed.     

Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences.

We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma.

Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DR1 region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNAs, including the human autonomously replicating sequence 1 (ARS1).     

Structural rearrangement of integrated hepatitis B virus DNA as well as cellular flanking DNA is present in chronically infected hepatic tissues.

Cellular DNAs from human livers chronically infected with hepatitis B virus (HBV) were analyzed by Southern blot hybridization for the presence of integrated HBV DNA. In 15 of 16 chronically infected hepatic tissues, random HBV DNA integration was evident. By molecular cloning and structural analyses of 19 integrants from three chronically infected hepatic tissues, deletion of cellular flanking DNA in all cases and rearrangement of HBV DNA with inverted duplication or translocation of cellular flanking DNA at the virus-cell junction in some cases were noted. Thus, the rearrangement of HBV DNA or cellular flanking DNA is not a specific incident of hepatocellular carcinoma formation. Detailed analyses of various integrants bearing rearranged viral DNA failed to indicate any gross structural alteration in cellular DNA, except for a small deletion at the integration site, indicating that viral DNA rearrangement with inverted duplication possibly occurs before integration of HBV DNA. Based on nucleotide sequencing analyses of virus-virus junctions, a one- to three-nucleotide identity was found. A mechanism for this inverted duplication of HBV DNA is proposed in which illegitimate recombination between two complementary viral strands may take place by means of a nucleotide identity at the junction site in a weakly homologous region (patchy homology) on one side of adjoining viral sequences. For virus-cell junctions, the mechanism may be basically similar to that for virus-virus junctions.     

Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells

Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.     

Cloned mouse mammary tumor virus DNA exhibits glucocorticoid-dependent expression in simian virus 40-transformed mink cells.

Mink lung epithelial cells were transfected with two cloned mouse mammary tumor virus (MMTV) DNAs, a 9-kilobase clone derived from an unintegrated exogenous viral genome and a 14-kilobase clone containing an integrated endogenous provirus along with cellular flanking sequences. Mink lung cells were chosen because they do not contain endogenous MMTV sequences. On the basis of our observation that simian virus 40 DNA efficiently transforms these cells, we isolated cell clones containing MMTV DNA by using transformation with simian virus 40 DNA as a selective marker in cotransfection experiments. Levels of the 9-kilobase MMTV mRNA representing the entire viral genome and of the spliced 4.4-kilobase mRNA which codes for the viral envelope proteins were glucocorticoid dependent in transformed cells. Expression of low levels of Pr77gag, the precursor of the group-specific viral core proteins, and of gPr73env, the precursor of the viral envelope proteins, was also hormone dependent. We conclude that these cloned MMTV DNAs contain all the information necessary for the synthesis of normal viral RNAs and proteins. These findings also provide further evidence that the DNA sequences involved in the hormone responsiveness of MMTV expression are contained within the viral genome.     

Isolation and analysis of retroviral integration targets by solo long terminal repeat inverse PCR

Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.