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Replication of retroviruses and retrotransposons depends on selecting a favorable chromosomal site for integration of their genomic DNA. Different retroelements meet this challenge by targeting distinctive chromosomal regions. Despite these differences, recent data hints at a common targeting mechanism-tethering of integration complexes to proteins bound at favorable sites.
Gene mapping experiments show that Fis-1, a preferred integration region for Friend murine leukemia virus in hematopoietic neoplasms, is extremely closely linked to Int-2, a preferred integration region for mouse mammary tumor virus in mammary carcinomas. Studies at the RNA and DNA level prove that these loci are distinct.
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The BL/VL(3) Kaplan radiation leukemia virus (RadLV-VL(3)) is a nondefective retrovirus that induces T cell lymphomas in several strains of mice. By using DNA probes derived from RadLV/VL(3) provirus-flanking sequences cloned from the BL/VL(3) cell line, we identified a DNA region rearranged in 5 of 19 tumors analysed (25%). All proviruses were integrated in the same 5'-to-3' orientation in a small DNA region called Kis1 (Kaplan integration site 1). This region was localized on distal mouse chromosome 2 in a region not previously identified as important to lymphomagenesis. The cells rearranged at the Kis1 locus represent a clonal subpopulation of the clonal tumor masses examined, indicating a probable role of Kis1 in tumor progression.
The integral membrane proteins of Sendai virus, the fusion protein F (Mr = 65,000) and the haemagglutinin-neuraminidase protein HN (Mr = 68,000), were used as a model protein mixture. They were subjected to size-exclusion high-performance liquid chromatography on Superose 6HR columns with eluents containing various additives in order to solubilize the proteins. The effect of the additives on the structure of the membrane proteins was investigated with conformation-dependent monoclonal antibodies, either directed against F or HN protein, and by determination of the haemagglutinating capacity of the HN protein. The results show that the structure of the HN protein is more easily disturbed by eluents than that of the F protein. When the elution conditions are mild, e.g., 0.1% octylglucoside, the structure of both proteins is conserved but no separation is obtained. Elution with a buffer containing 0.05% sarkosyl (dodecyl methylglycine sodium salt) did not affect the structure and resulted in pure F protein. Pretreatment of the Amberlite XAD-2-treated Sendai virus envelope extract with 4% sodium dodecyl sulphate (SDS) and elution with 0.1% SDS in 50 mM sodium phosphate (pH 6.5) altered the structure of the HN protein but resulted in purification of the tetramer and the dimer of the HN protein, and the monomer of the F protein.
To replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. Integration is not random in the host genome but favors particular regions, and preferences differ among retroviruses. Several mechanisms might play a part in this favored integration targeting: (i) open chromatin might be preferentially accessible for viral DNA integration; (ii) DNA replication during cell division might facilitate access of integration complexes to favored sites; and (iii) cellular proteins bound to the host chromosome might tether integration complexes to favored regions. This review summarizes recent advances in understanding the mechanisms of retroviral integration, focusing on LEDGF/p75--the first cellular protein shown to have a role in directing HIV DNA integration. Studies on LEDGF/p75 indicate that it directs HIV integration site selection by a tethering interaction, whereas the chromatin accessibility or cell cycle models are less well supported. Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy.
We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.
The integration sites for viral DNA in cells infected with Moloney murine leukemia virus (M-MuLV) were studied by restriction endonuclease cleavage of cellular DNA followed by electrophoresis in agarose gels, blot transfer to nitrocellulose, and detection by M-MuLV-related sequences by hybridization with high-specific-activity 32P-labeled M-MuLV complementary DNA. When EcoRI was used to cleave cellular DNA, numerous DNA fragments with sequence homology to M-MuLV were detected in uninfected mouse cell DNA. These endogenous sequences are mouse specific since they are not detectable in rat cell DNA, and are related to the 38S genomic RNA of M-MuLV. Infected cells contain additional M-MuLV-specific DNA fragments which are not detected in uninfected cells. Different patterns of M-MuLV-specific DNA fragments were detected in each cloned infected line examined. These data suggest the existence of multiple sites for integration of M-MuLV DNA in infected mouse fibroblasts. Cleavage of infected cell DNA with BamHI, which cleaves M-MuLV viral DNA at least twice, released the internal BamHI B fragment from each infected line, confirming the presence of integrated M-MuLV DNA sequences in each infected cell line which retain some features of the sequence organization of unintegrated M-MuLV DNA.
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The INT6 gene is a common integration site for the mouse mammary tumor virus in mouse mammary tumors. We have determined that the human homolog of INT6 is located on chromosome region 8q22-q23. A processed INT6 pseudogene is located on chromosome 6q. INT6 is composed of 13 exons that span 45 kb of genomic DNA. The deduced amino acid sequence of the gene product is identical to the mouse protein and contains three potential translation start signals. We have examined 100 primary breast carcinoma DNAs for evidence of genetic alteration affecting INT6. Loss of heterozyosity (LOH) was detected in 11 of 39 (28%) of the tumor samples informative for a polymorphic sequence in intron 7 of INT6. Since single-strand conformation and hybrid mismatch analysis of the remaining allele in these tumor DNAs failed to detect any mutations, we conclude that the target gene for LOH must be closely linked to INT6.
We report here the design, construction and testing of a self-inactivating (Sin) retrovirus promoter-trap vector suitable for identifying and isolating transcriptionally active regions from the mouse genome. When this vector, which contains the bacterial aph gene as its reporter, is integrated into a site downstream from an active host cell promoter, it expresses aph, whose product, aminoglycoside phosphotransferase, produces resistance to the antibiotic G418 in mammalian cells. The construct also contains a native aph promoter which functions in bacteria, but not in mouse cells, to express kanamycin (Km) resistance, plus an adjacent pBR322-derived replication origin. Thus, mammalian DNA segments containing actively transcribed regions flanking aph can be quickly isolated by restriction endonuclease treatment of total DNA from provirus-containing mouse cells, followed by self-ligation, transformation and Km selection of plasmids carried by bacteria transformed with this DNA. We tested this Sin retrovirus promoter-trap system by isolating eight DNA segments upstream to the provirus integration sites in the genome of virus-infected mouse F9 cells. We found that the Sin retrovirus vector produces a high yield of infectious virus particles carrying aph, and that the isolated genomic DNA fragments of F9 cells are transcriptionally active.
Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, we further show that blastoderms can be infected in culture and in ovo. In ovo, lacZ expression is seen within 24 hr of virus inoculation, and by 96 hr stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.
The integrated proviral genome of Rauscher murine leukemia virus was molecularly cloned in a bacteriophage Charon 4A vector after the proviral sequences were enriched by sequential RPC-5 column chromatography and sucrose gradient centrifugation. A recombinant DNA clone, lambda-RV-1, possessing a 12-kilobase-pair EcoRI insert, was shown to contain the entire 8.8-kilobase-pair leukemia virus genome flanked by rat cellular sequences at the 5' and 3' ends. This DNA fragment was biologically active, inducing the release of virion-associated reverse transcriptase activity with as little as 10 ng of DNA insert. The virus induced XC plaque formation at high titers on NIH/3T3 and BALB/3T3 cells and demonstrated identity with the parental virus in radioimmunoassays for the highly type-specific gag gene-coded p12 protein. The molecularly cloned Rauscher murine leukemia virus should be useful in studying the molecular mechanisms involved in the transformation of specific lymphoid target cells by chronic mouse leukemia viruses.
AIM AND BACKGROUND: Hepatitis B virus is implicated in the development of hepatocellular caracinoma. No oncogenes have been identified within the viral genome. Furthermore, it frequently fragments after integration into the hepatocyte genome. Simultaneous investigations of hepatitis B virus integration patterns and genetic changes in precancerous tissues are important to understand the role played by hepatitis B virus integration in hepatocellular caracinoma. METHOD: We used a combination approach of dual characterization of highly polymorphic loci and the change in hepatitis B virus-DNA integration pattern. Large regenerative nodules were dissected from 6 explanted hepatitis B virus infected cirrhotic livers. Nodules within each liver segment were schematically mapped and histopathologically analyzed. Genomic DNA from each nodule was analyzed for hepatitis B virus integration and the genetic stability of 12 microsatellite loci including D3S2321, D8S1022, D17S1159, D4S2281, D5S1/2, D16S675, D16S685, D16S490, D16S526, D16S673, D16S677 and D16S690. RESULTS: Data from different liver segments revealed few viral integrations and average allele loss. The most exciting results came from a segment containing a set of clonally and spatially related nodules having similar histologic features, a progressive lineage of allele loss, HBV integration and loss of integration. CONCLUSIONS: This model portrait, a scenario of genetic events that precede tumor formation where the acquisition and loss of hepatitis B virus integrations in clonally related regenerative nodules, might explain how the virus acts as a hit-and-run mutagen.
To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host. The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified. Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system. Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1. We find that many inhibitors active against purified integrase are inactive against PICs. Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase. We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.
AIMS: Myeloblastosis Associated Virus type 1 (N) [MAV 1(N)] induces specifically nephroblastomas in 8-10 weeks when injected to newborn chicken. The MAV-induced nephroblastomas constitute a unique animal model of the pediatric Wilms' tumor. We have made use of three independent nephroblastomas that represent increasing tumor grades, to identify the host DNA regions in which MAV proviral sequences were integrated. METHODS: Cellular sequences localized next to MAV-integration sites in the tumor DNAs were used to screen a Bacterial Artificial Chromosomes (BACs) library and isolate BACs containing about 150 kilobases of normal DNA corresponding to MAV integration regions (MIRs). These BACs were mapped on the chicken chromosomes by Fluorescent In Situ Hybridization (FISH) and used for molecular studies. RESULTS: The different MAV integration sites that were conserved after tumor cell selection identify genes involved in the control of cell signaling and proliferation. Syntenic fragments in human DNA contain genes whose products have been involved in normal and pathological kidney development, and several oncogenes responsible for tumorigenesis in human. CONCLUSION: The identification of putative target genes for MAV provides important clues for the understanding of the MAV pathogenic potential. These studies identified ADAMTS1 as a gene upregulated in MAV-induced nephroblastoma and established that ccn3/nov is not a preferential site of integration for MAV as previously thought. The present results support our hypothesis that the highly efficient and specific MAV-induced tumorigenesis results from the alteration of multiple target genes in differentiating blastemal cells, some of which are required for the progression to highly aggressive stages. This study reinforces our previous conclusions that the MAV-induced nephroblastoma constitutes an excellent model in which to characterize new potential oncogenes and tumor suppressors involved in the establishment and maintenance of tumors.
Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory. We observe here that integration of a retrovirus from the spumavirus family depends mainly on the quantity of viral DNA produced. Moreover, we found that IN directly participates to linear DNA production from 2-LTR circles by specifically cleaving the conserved palindromic sequence found at LTR-LTR junctions. These results challenge the prevailing view that integrase essential function is to catalyze retroviral DNA integration. Integrase activity upstream of this step, by controlling linear DNA production, is sufficient to explain the absolute requirement for this enzyme. The novel role of IN over 2-LTR circle junctions accounts for the pleiotropic effects observed in cells infected with IN mutants. It may explain why 1) 2-LTR circles accumulate in vivo in mutants carrying a defective IN while their linear and integrated DNA pools decrease; 2) why both LTRs are processed in a concerted manner. It also resolves the original puzzle concerning the integration of spumaretroviruses. More generally, it suggests to reassess 2-LTR circles as functional intermediates in the retrovirus cycle and to reconsider the idea that formation of the integrated provirus is an essential step of retrovirus production.