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DNA Recombination Is Sufficient for Retroviral Transduction JODY R Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2460-2464, March 1995 Biochemistry DNA recombination is sufficient for retroviral transduction JODY R. SCHWARTZ, Susi DUESBERG, AND PETER H. DUESBERG Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3206 Contributed by Peter H. Duesberg, November 28, 1994 ABSTRACT Oncogenic retroviruses carry coding se- quences that are transduced from cellular protooncogenes. Nat- env pOl X ural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentall, its mechanism has been studied in terms oftwoehypotheses: (i) the DNAmodel,which postulates two ___ _-I ____ DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. proto-onc gene Here we use two viral DNA constructs to test the prediction ofthe DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromo- somal protooncogene coding region. For the DNA model to be ga-onc - gag viable, such recombinant viruses must be infectious without the w5 essential tract that precedes the 3' purportedly polypurine (ppt) FIG. 1. The DNA model of retroviral transduction. The model long terminal repeat (LTR) of all retroviruses. Our constructs proposes that the 5' retrovirus/protooncogene junction is achieved by consist ofa ras coding region from Harvey sarcoma virus which nonhomologous recombination between a circular provirus with a is naturally linked at the 5' end to a retroviral LTR and single LTR. Such proviruses are common in virus-infected cells (1). artificially linked at the 3' end either directly (construct NdN) or The 3' protooncogene/retrovirus recombination is proposed to result by a cellular sequence (construct SU) to the 5' LTR of a from the conventional integration of circular or linear provirus with retrovirus. Both constructs lack the ppt, and the LTR of NdN two LTRs. U3, segment of nucleotides which is unique to the 3' end; even lacks 30 nucleotides at the 5' end. Both constructs proved US, segment of nucleotides which is unique to the 5' end; ga-onc, to be infectious, producing viruses at titers of 105 focus-forming postulated fusion gene arising from recombination between retroviral units per ml. Sequence analysis proved that both viruses were gag and cellular protooncogene. colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA integrated, because they lack a standard integration site, which recombination is sufficient for retroviral transduction and that is defined by two adjacent LTRs (1, 2). Since normal provi- neither the ppt nor the complete LTR is essential for retrovirus ruses integrate between two adjacent LTRs, an aberrant replication. DNA recombination explains the following observa- provirus with a single LTR would have to integrate randomly tions by others that cannot be reconciled with the RNA model: (i) with regard to the viral genome, thus generating, among experimental transduction is independent of the packaging others, 5' retroviral-3' protooncogene hybrid genes (Fig. 1) efficiency ofviral RNA, and (ii) experimental transduction may (3). invert sequences with respect to others, as expected for DNA The DNA model and the RNA model differ only with regard recombination during transfection. to the 3' protooncogene/virus recombination. The RNA model suggests that the 3' protooncogene/virus recombination occurs All oncogenic retroviruses carry an internal coding sequence by copy choice during reverse transcription between retroviral transduced from one of a group of cellular genes, termed RNA and protooncogene mRNA (1, 4-7). The DNA model protooncogenes. Natural transduction by means of illegitimate suggests that the 3' recombination occurs between viral and cell recombination between viral and cellular genomes cannot be DNA (3, 8, 9). It predicts that the 3' protooncogene/provirus experimentally reproduced, because it is extremely rare. Only recombination could result from the integration of a complete about 50 cases have been observed in the long history of retrovirus 3' ofthe coding region of a protooncogene (Fig. 1) (3). retrovirus research (1). There are, however, experimental In this case the 5' LTR ofthe integrating provirus would function models of transduction, termed the DNA model and the RNA as the 3' LTR of the resulting recombinant virus (Fig. 1). model, derived from two competing hypotheses for how ret- This mechanism has one potential limitation. The resulting roviruses transduce these coding sequences from cellular recombinant virus would lack a short, retroviral sequence protooncogenes. immediately prior to the 3' LTR, termed the polypurine tract Both models assume that the 5' virus/protooncogene re- (ppt), which is thought to be essential for plus-strand viral combination occurs at the DNA level-i.e., between cellular DNA synthesis (1, 10, 11). This sequence would be missing DNA and retroviral DNA, also termed proviral DNA (which from the inifial recombinants predicted by this mechanism is transcribed from viral RNA). One interpretation of this because the shared LTR would be linked directly to the 3' model predicts that the 5' recombination with a protoonco- region of a protooncogene DNA sequence (Fig. 1). Thus, for gene results from random integration of a DNA provirus with the DNA model to be viable, retroviruses must be able to a single virus long terminal repeat (LTR; Fig. 1). Aberrant replicate without a ppt. proviruses with single LTRs are abundant in all retrovirus- Here, we have tested the prediction of the DNA model that infected cells, but they are neither effectively nor specifically the 3' DNA recombination is achieved by conventional inte- gration of a retroviral DNA 3' of the protooncogene coding The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: ppt, polypurine tract; LTR, long terminal repeat; accordance with 18 U.S.C. §1734 solely to indicate this fact. HaSV, Harvey sarcoma virus. 2460 Downloaded by guest on September 25, 2021 Biochemistry: Schwartz etal. Proc. Nati Acad Sci USA 92 (1995) 2461 region by using two retroviral DNA constructs that lack the generated to test the 3' DNA recombination predicted by the ppt sequence. These constructs include an oncogenic Harvey DNA model (Fig. 1) and analyzed for their ability to form sarcoma virus (HaSV) rascoding region, which is naturally replicating viruses. Both are derivatives of murine HaSV and linked at the 5' end to a retroviral LTR. At the 3' end, the ras would be expected to transform murine cells in culture (13, coding region of our construct is artificially linked either 14). Both viral constructs lack the3'-terminal ppt that maps directly or by means of a cellular sequence to the 5' LTR of a just upstream of the 3' LTR of retroviruses (Fig. 2) (1). NdN retrovirus (Fig. 2). Upon transfection into mouse cells, both also lacks the 5' 30 bases of the U3 region in both LTRs (1), constructs proved to generate sarcomagenic viruses, replicat- which are thought to be essential for provirus integration (2). ing at titers of105 focus-forming units in the presence of helper Since both of these elements are considered essential for virus virus. The results indicate that DNA recombination can be replication (1, 2), the transduction intermediates predicted by sufficient for retroviral transduction. the DNA model may not be viable. Thus, testing their ability to replicate is crucial for our model. Generation of Viruses from Proviral Constructs NdN and MATERIALS AND METHODS SU Predicted by the DNA Model. NdN virus. The most efficient The SU provirus was synthesized from a molecularly cloned method of recovering infectious virus from proviral DNA of Harvey sarcoma provirus, termed pH1/RSNhe (8), and a transforming retroviruses that lack essential structural genes molecul' rlycloned murine retrovirus, termed AKRvirus (12). (1), like HaSV, involves simultaneous transfection with Molo- pHi /R5Nhe differs from the complete Harvey sartoma pro- ney murine helper virus DNA (13). This is because over 99% virus by a deletion that extends from the 5' end of the 5' LTR of the transfected DNA that enters the cell is not integrated in to 'an EcoRV site 215 nt downstream (8). The deletion is the cellular chromosome but survives as a cellular plasmid for restricted to the nontranscribed region of the LTR and does several cell generations (13). Such unintegrated DNA is not affect the ability of the provirus to transform cells (8). To transcribed like integrated DNA but is converted to infectious generate SU provirus, the 4.5-kb region of pH1/R5Nhe virus only if a helper proviral plasmid is simultaneously I transcribed in the same cell. The genome of the defective extending from the Sal site in pBR322 to an Nhe I site 3' of transforming virus will then be packaged into helper virus ras was ligated with a 2.4-kb region of pAKR 623 provirus protein capsids and will thus be able to infect cells. extending from a cellular Nhe I site 5' of the viral LTR to a Sal Therefore, a semiconfluent culture of mouse C3H10T1/2 I site within the viralgag gene (Fig. 2) (12). The resulting 6.9-kb cells in a 10-cm Petri dish was simultaneously transfected with plasmid carried the SU recombinant provirus depicted in Fig. 5,g of molecularly cloned NdN provirus and ,ug1 of cloned 2. helper Moloney murine provirus as described (13, 14). Five The NdN provirus was synthesized in two steps. In the first days later the culture was split into four dishes, and 15 days step, a 0.8-kb DNA fragment of Harvey sarcoma provirus (13) later 20 large and about 20 smaller foci of transformed cells extending from the Nhe I site in the 5' viral LTR at nucleotide had appeared.
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