Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites

Rat hepatoma cells infected with mou-se mammary tumor virus (MMTV) acquire viral DNA that becomes covalently linked to the cell DNA. Using restriction endonucleases and the DNA transfer procedure of Southern [Southern, E. M. (1975) Glucocorticoid hormones (e.g., dexamethasone) selectively stimulate the production of mouse mammary tumor virus (MMTV) in both mouse mammary carcinoma cells (1-7) and heterologous cells infected with MMTV (8,9). This stimulation of MMTV gene expression appears to occur via a rapid increase in the rate of synthesis of viral RNA (10,11). Results of earlier experiments suggest that the template for viral RNA synthesis is MMTV DNA that is covalently linked to the cellular DNA (i.e., proviral DNA) (12). When individual clones of MMTVinfected rat hepatoma cells (HTC) were tested, molecular hybridization assays revealed the presence of various numbers of MMTV proviruses in these cells. Moreover, the amounts of viral RNA and the magnitude of the hormonal response also varied widely among the clones, but not in a fashion that was directly related to proviral DNA copy number (13,14).The observation that MMTV gene expression is not simply a function of the dosage of viral DNA sequences in these cells prompted us to investigate the structure and organization of the viral sequences that are integrated into the host genome. Several questions were of particular interest: Can many sites within the host genome accommodate proviruses? In clones containing multiple copies of viral DNA, are the proviruses integrated in tandem relative to one another? Is a unique (or restricted) region of the viral genome utilized for integrative recombination?We have approached these questions by cleaving infected cell DNAs with restriction endonucleases, separating the DNA fragments electrophoretically, and identifying fragments containing viral sequences by molecular hybridization; these methods have been discussed in detail in analyses of other viral and cellular genes (15)(16)(17)

show abstract

“…Results similar to those described here concerning the sites of integration of retrovirus DNA have recently been described (31)(32)(33).…”

Section: Discussionsupporting

confidence: 73%

Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells.

Ringold¹,

Shank²,

Varmus³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

110

View full text Add to dashboard Cite

show abstract

“…Integration can occur into most sites of the chromosome (11)(12)(13). However, certain sites within a genome can be used at a frequency several hundred-fold greater than chance (11,14,15).…”

mentioning

confidence: 99%

Role of the Nonspecific DNA-binding Region and α Helices within the Core Domain of Retroviral Integrase in Selecting Target DNA Sites for Integration

Appa¹,

Shin²,

Lee³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Retroviral integrase plays an important role in choosing host chromosomal sites for integration of the cDNA copy of the viral genome. The domain responsible for target site selection has been previously mapped to the central core of the protein (amino acid residues 49 -238). Chimeric integrases between human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) were prepared to examine the involvement of a nonspecific DNA-binding region (residues 213-266) and certain ␣ helices within the core domain in target site selection. Determination of the distribution and frequency of integration events of the chimeric integrases narrowed the target site-specifying motif to within residues 49 -187 and showed that ␣3 and ␣4 helices (residues 123-166) were not involved in target site selection. Furthermore, the chimera with the ␣2 helix (residues 118 -121) of FIV identity displayed characteristic integration events from both HIV-1 and FIV integrases. The results indicate that the ␣2 helix plays a role in target site preference as either part of a larger or multiple target site-specifying motif.

show abstract

“…In all likelihood, these differences are due to genetic variation in the viral genome, a situation already well known in other systems (21)(22)(23). Alternative explanations for these differences might be the use of different regions of the proviral sequence in the integration into the genome of the two animals or the insertion of cellular DNA sequences into the provirus.…”

Section: Discussionmentioning

confidence: 97%

“…Alternative explanations for these differences might be the use of different regions of the proviral sequence in the integration into the genome of the two animals or the insertion of cellular DNA sequences into the provirus. The first explanation would, however, lead to noncolinearity of proviral DNA and viral RNA (17), a possibility recently disproven in other retrovirus systems (21,24). In contrast, the proviral sequences found in the DNAs of animals 92 and 928 might be identical, because they seem to share an internal proviral fragment of Mr 2.4 and 106.…”

Section: Discussionmentioning

confidence: 99%

Integration of bovine leukemia virus DNA in the bovine genome.

Kettmann¹,

Meunier–Rotival²,

Cortadas³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

ABSTRACrDNA preparations from circulating leukocytes, lymph node tumors, and spleens of three bovine leukemia virus-infected cattle were fractionated by Cs2SO4/3,6-bis(acetatomercurimethyl)dioxane density gradient centrifugation. Bovine leukemia virus proviral sequences were found in large GC-rich fragments having a buoyant density in CsCl close to 1.708 g/cm3. Provirus integration, therefore, does not take place at random locations in the host genome, but in a specific class of DNA segments. Hybridization of cDNA synthesized on viral RNA to EcoRI and Xba [restriction fragments of the DNA from infected cells showed that: (i) only one copy of proviral DNA is integrated per haploid genome; (ii) different restriction patterns were found in the proviral DNAs present in the genomes of different animals, providing evidence for the existence of several viral strains or mutants; and (iii) different integration sites for the proviral DNA were found in the genome of different animals and of different infected cells in the same animal. The latter finding strongly suggests a polyclonal origin of bovine leukemia virus-infected cells.Bovine leukemia virus (BLV), an exogenous retrovirus (1-3) of cattle, is the pathogenic agent of enzootic bovine leukosis. The target cell is the B lymphocyte (4). BLV infection may induce persistent lymphocytosis (characterized by an increased number of apparently normal B lymphocytes in the peripheral blood of the infected animal) and, in a later stage, lymph node tumors.In the present work, we have tried to answer a number of questions concerning the integration process of proviral DNA into the host cell genome-namely: (i) whether integration occurs at random in the bovine genome or in one of the DNA components in' which this genome can be resolved by density gradient centrifugation techniques (5-7); (ii)'how many proviral copies are integrated in the-genome; (iii) whether integration occurs at the same chromosomal location in the genomes of different infected animals and in the genomes of different infected cells in the same animal; and (iv) whether different viral strains or mutants can be detected by studying the proviral sequences.MATERIALS AND METHODS Bovine Tissues and Cells. Bovine material was collected from two field cases of enzootic bovine leukosis (animals 15 and 928) and from an experimentally infected animal (animal 92); in the latter case, the BLV was obtained from the infected herd to which animal 928 belonged. Animals 15 and 92 were at the tumoral stage of the disease with splenomegaly (8). Animal 928 was in persistent lymphocytosis without tumors (8). Leukocytes (W15 and W928), two lymph node tumors (T15 and T92), and spleens (S1S and S92) were used as sources of DNA. The thymus (CT) from a normal calf was used as a source of control DNA.Preparation of Virus and Viral RNAs and cDNA Synthesis. BLV production and purification and viral RNA extraction and purification were carried out as described (9). The two poly (A+) 38S RNAs present in each viral particle were prepared from ...

show abstract

Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites

Cited by 344 publications

References 33 publications

Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells.

Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells.

Role of the Nonspecific DNA-binding Region and α Helices within the Core Domain of Retroviral Integrase in Selecting Target DNA Sites for Integration

Integration of bovine leukemia virus DNA in the bovine genome.

Contact Info

Product

Resources

About