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 sequences 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 integration 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. position 5093 (1) to a BamHI site between the LTR and ras was Eight of these foci were picked and grown as clonal cultures ligated into the Nhe I and BamHI sites of pBR322. In a second for analysis of proviral DNAs. For this purpose total cell DNA step, the resulting plasmid was linearized at the Nhe I site and of clonal cultures was digested with the restriction enzyme Nhe ligated to a 2.1-kb fragment from Harvey sarcoma provirus I, resolved by electrophoresis through 1% agarose, transferred extending from the Nhe I site in the 5' LTR to the Nhe I site to nitrocellulose, and hybridized with 32P-labeled ras DNA as 3' of ras at nucleotide position 1693 (Fig. 2) (1). The resulting described (15). As can be seen in Fig. 3, each transfected and plasmid with the two truncated LTRs arranged in the same transformed culture contained an electrophoretically distinct orientation was selected and termed NdN. ras DNA (Fig. 3, Provirus, lanes 1-8) and shared with normal C3H1T1/2 cells (Fig. 3, Provirus, lanes 9 and 10) 4-kb and 1-kb RESULTS Nhe I proto-ras DNA restriction fragments. Only one of eight focus-derived clonal cultures, termed clone 8, contained ras- Construction of Proviral Transductants Predicted by the specific DNA with the same electrophoretic mobility as Nhe DNA Model. Here, we have used proviral DNA constructs to I-digested input NdN provirus DNA (Fig. 3, Provirus, lane 8). test the prediction of the DNA model that the 3' recombina- The probable reason for the structural heterogeneity of ras- tion in transduction can be achieved by conventional retrovirus specific proviral DNAs in transformed clonal cultures is integration. Two proviral constructs, NdN and SU, have been recombination between DNAs prior to integration-i.e., between NdN and Moloney murine provirus plasmids, as well as Nhe RV Nhe carrier DNA (8, 16). Since only clone 8 contained Nhe I-resistant, ras-positive, HaSV U. _ proviral DNA of the size expected from the input NdN E . provirus, namely 2.2 kb, the medium from this clonal culture was IV Nhe analyzed for the presence of NdN virus that would replicate if aided by helper virus. For this purpose, the medium of clone 8 SU cultures was added to cultures of mouse C3H1OT/2 cells. Five to seven days later the C3H1OT1/2 cells were transformed. The transforming NdN virus in the culture medium was titered Nhe Nhe by a serial end-point dilution as described (14). The titer virus was 1kb NdN of C3H1T/2 cell-transforming units of the NdN 5_ i~~~ 3 x 105 (Table 1). The other clones were not analyzed for transforming viruses because the ras-positive proviruses FIG. 2. Genetic structures of Harvey sarcoma provirus and two were recombinants that may have recovered a ppt and other recombinant viruses, SU and NdN, constructed to test the 3' recom- retrovirus-specific sequences from the helper Moloney mu- bination of transduction according to the DNA model (Fig. 1). In the rine provirus. SU construct the 3' LTR of a retrovirus is linked by means of a cellular DNA sequence to the ras coding region of Harvey sarcoma provirus. SUvirus. In view of the high rate of recombination observed In the NdN construct the 3' LTR of a retrovirus is linked from an between defective transforming and helper proviral DNAs internal Nhe I restriction site, 30 nucleotides within the U3 region of upon simultaneous transfection, the SU provirus was intro- the LTR, to the ras coding region of Harvey sarcoma provirus. duced into C3H1OT1/2 cells in the absence of helper virus DNA. Downloaded by guest on September 25, 2021 2462 Biochemistry: Schwartz et al. Proc. NatL Acad Sci. USA 92 (1995)

Provirus Virus generated by the synthetic, ppt-free proviruses NdN and SU 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 were analyzed to determine whether the transforming viruses Origin - had retained the structure of the input proviruses or had derived a ppt from the helper virus by recombination. NdN virus. The genetic structure of the NdN virus was 23 - investigated as proviral DNA in virus-transformed C3H10T1/2 9.3 - cells. For this purpose the DNA of NdN virus /Moloney 6.6- murine leukemia virus complex-transformed cells was digested with Nhe I, electrophoresed in agarose, and hybridized with 32P-labeled ras DNA (Fig. 3, Virus), as described above. The DNAs of simian virus 40-transformed C3H10T1/2 cells, NdN 2.2- 2.0 - provirus-transformed clone 8 cells, and the bacterial plasmid carrying cloned NdN provirus, pNdN, were analyzed in par- allel after digestion with Nhe I under the same conditions. As can be seen in Fig. 3, digestion of DNA from cells infected with the NdN virus produced a provirus DNA of 2.2 kb (Fig. 3, Virus, lane 2) that was indistinguishable in size from the synthetic NdN provirus cloned in the bacterial plasmid (Fig. 3, 0.6 - Provirus, lanes 11 and 12) and from the NdN provirus in clone 8 cells (Fig. 3, Provirus, lane 8 and Virus, lane 3). All three FIG. 3. ras-containing, transforming proviruses in mouse C3H10T1/2 C3H10T1/2 cultures also contained the 4-kb and 1-kb Nhe I cells transfected by NdN provirus. C3H1OT½/2 cells were simultaneously proto-ras restriction fragments characteristic of uninfected transfected with cloned plasmids carrying NdN provirus and Molo- cells (Fig. 3, Virus, lane 1). ney murine leukemia provirus. Foci of transformed cells were SU virus. The nucleic acid of the SU virus/Moloney murine selected and grown up into mass cultures. The DNA of eight clonal leukemia virus complex was first investigated as RNA ex- cultures was digested with Nhe I and resolved by electrophoresis tracted from virions produced by three clonal cultures of SU through 1% agarose. After transfer of the DNA to nitrocellulose, provirus-transformed, nonproducer cells described above, the ras DNA was identified by hybridization with 32P-ras DNA and which were then superinfected with Moloney murine leukemia autoradiography as described (15). Provirus, lanes 1-8, clonal virus (see Table 1) and then as RNA from seven clonal cultures cultures of NdN provirus and Moloney murine leukemia provirus- transformed cells; lanes 9 and 10, uninfected C3H10T1/2 cells; lanes of virus-infected cells. The virus produced by each of these 11 and 12, Nhe I-digested plasmids carrying NdN provirus. Virus, cultures was harvested, and the RNA was extracted, poly(A)- lane 1, uninfected C3H10T½/2 cells; lane 2, C3H10T1/2 cells infected selected, and electrophoresed as described (15). As can be seen with virus produced by cell clone 8 (Provirus; lane 8); lane 3, same as in Fig. 4, the ras-specific SU virus RNA produced by each of Provirus, lane 8. The positions ofHindIII-digested A DNA fragments are three provirus-transformed cultures and by six of seven virus- identified at the left in kb. transformed cultures measured about 2.8 kb (Fig. 4, Provirus, lanes 1-3, and Virus, lanes 1-7). This is the expected RNA size, About 10 ,ug of plasmid carrying SU provirus and 10 jig of calf considering that the provirus measures 2.6 kb (Fig. 1) and that thymus carrier DNA were transfected as described above. Two retroviral RNAs carry a 200-residue poly(A) tract at their 3' days after transfection the culture was confluent. It was split ends (17). 1:1 and allowed to reach confluence again. As expected, the In addition to the 2.8-kb RNA, each of these six viral clones yield of stable transformants in the absence of helper virus was contained a minor RNA component of about 3 kb, the origin very low (13, 14), only about nine foci per 10 ,ug of DNA 3 of which is not clear. However, one ras-containing virus from weeks after transfection. an SU provirus-transformed clonal culture contained a unique Clonal cultures derived from three foci of SU provirus- RNA of over 3 kb, probably from a recombinant provirus (Fig. transformed cells were superinfected with Moloney murine 4, Virus, lane 3). This indicates that recombination during leukemia virus. After incubation for 1 week, each super- transfection between transforming provirus and helper pro- natant was used to inoculate C3H1OT1/2 cells. About 5 days virus, as had been observed during simultaneous transfection later, each virus-infected culture of C3H1OT1/2 cells was with NdN and Moloney murine leukemia provirus DNAs, can transformed and the transforming virus in the medium was be significantly reduced by transfection in the absence of titered. The focus-forming titers of transforming SU virus helper provirus. produced by these clonal cultures were all around 105 per ml. On the basis of electrophoretic properties, it would appear The exact titer of one is shown in Table 1. that the 3' proto-ras/LTR recombinants NdN and SU pre- It follows that both NdN and SU proviruses are able to dicted by the DNA model are each able to replicate as viruses generate efficiently replicating transforming viruses, despite that appear to be isogenic with the corresponding proviral the absence of the 3'-terminal ppt and the 5' 30 nucleotides of constructs. the LTR ofNdN. The titers ofthe NdN and SU viruses are only NdN Virus Replicates Without a ppt Sequence and Without about 1/10th that of wild-type HaSV, the titer of which is the 30 5' Nucleotides of Viral LTR. Although the physical about 106 focus-forming units per ml (13). structures of the NdN and SU viruses corresponded exactly to Genetic Structures of the Transforming NdN and SU Vi- the input proviruses, it may be argued that they had recovered ruses. Next, the genetic structures of the transforming viruses ppt sequences and that NdN also had recovered its missing 30 5' LTR nucleotides by recombination with helper virus. Table 1. Focus titers of ppt-free SU and NdN To determine whether the sequence preceding the 3' LTR ras-containing viruses of the NdN virus was indeed the same as that of the input NdN Dilution of SU virus, no. of NdN virus, no. of provirus (Fig. 2), the 3' proviral region from NdN virus- virus stock foci per ml foci per ml transformed C3H10T1/2 cells was sequenced. For this purpose the proviral DNA of NdN virus-transformed cells was first 10-2 254 >250 1o-3 54 92 amplified by PCR using two NdN virus-specific primers. The 5 5' primer consisted of 25 nt corresponding to HaSV sequence 1o-4 18 positions 1255-1279, and the 3' primer consisted of 24 nt 10-5 1 3 corresponding to HaSV LTR sequences from position 5103 to Downloaded by guest on September 25, 2021 Biochemistry: Schwartz et al. Proc. Natl. Acad. Sci. USA 92 (1995) 2463 Provirus Virus Nhe I restriction sites at positions 1693 and 5093 (Fig. 2; ref.

1 2 3 1 2 3 4 5 6 7 1). As can be seen in Fig. 5, lane 2, the DNA fragment from NdN virus-transformed cells amplified with these primers had Origin - - - Oric Origin the expected size-i.e., it coelectrophoresed exactly with DNAs amplified from NdN provirus-transformed clone 8 cells

9.5 - 9.5 - -9.5 (Fig. 5, lane 1) and with DNA from the pNdN plasmid 7.5 - 7.5- -7.5 amplified with the same primers. It was about 100 nt smaller than the 560-nt A phage DNA marker (Fig. 5, lane 3). -4.4 4.4 - 4.4 - For further analysis, the PCR-amplified DNA of NdN virus-transformed C3H10T1/2 cells was digested with Fsp I,

-2.4 which cuts NdN provirus in the ras gene at the HaSV sequence 2.4- 9 2.4 - position 1290 (1) and with Nhe I, which cuts NdN provirus at -1.4 the ras/LTR border at HaSV sequence position 1693/5093 1.4 - 1.4 - (Fig. 2), to generate a 403-nt fragment. The expected 403-bp DNA was obtained (data not shown) and was cloned into the Pvu II and Nhe I sites of pBR322 for sequence analysis. Four clones, termed pFN1, -3, -5, and -7, were selected on the basis of the predicted sizes of two Pst I and Dra III restriction fragments of 0.9 and 1.9 kb and two HindIII and Bbs I restriction fragments of 2.4 and 0.4 kb (data not shown) (1). The sequences of the 206 3'-terminal NdN virus-specific nt FIG. 4. ras-positive RNAs from viruses produced by mouse of three of four clones were identical to the 206 nt that map C3H10T½/2 cells either transfected with the SU proviral construct (Fig. upstream of the Nhe I restriction site 3' ofthe ras gene ofHaSV 2) or infected with the SU/Moloney murine leukemia virus complex. at position 1693 (1). In one of the four clones, pFN1, there Provirus, lanes 1-3, ras-positive RNAs of SU virus produced by were five uncertainties at sites 188, 189, 194, 195, and 199 (Fig. provirus-infected cells. C3H1OT½/2 mouse cells were transfected with 6). SU provirus, and transformed foci were selected and grown into clonal It that the cultures. RNA was isolated from three such clones and analyzed on follows retroviruses can replicate without ppt denaturing agarose gels by hybridization with a ras-specific probe. sequence and without the 30-nt sequence from the 5' end of Three clonal, nonproducer cultures of SU-transformed C3H10T½/2 the LTR. mouse cells were each superinfected with Moloney murine leukemia virus. The RNA from the resulting virus-infected cells was poly(A)- selected, electrophoresed through 2 M formaldehyde/1% agarose DISCUSSION gels, and transferred to nitrocellulose. After hybridization with 32p- The DNA Model Is More Compatible with Retroviral Trans- labeled ras DNA, ras RNA was identified by autoradiography as duction than Is the RNA Model. Our results lend direct support described (15). Virus, lanes 1-7, ras-positive 'RNAs of SU virus to the DNA model of transduction but do not exclude the RNA produced by SU virus-infected cells. Viruses were harvested from seven SU provirus-transformed, nonproducer cultures super- model. The RNA model has been favored by retrovirologists infected with Moloney murine leukemia virus. C3H10T1/2 cells were despite its intrinsic asymmetry-i.e., a 5' DNA and a 3' RNA infected with each of the seven virus stocks and the ras RNAs of recombination (1, 5-7). This bias for the RNA model was the resulting viruses were analyzed as described above. The positions originally based on experiments which suggested "efficient ille- of RNA molecular size standards are identified at the margins in kb. gitimate recombination" during reverse transcription (4). In these experiments, cells transformed by a Harvey sarcoma provirus 5126 (1). In NdN virus, these sequences are separated by 471 that lacked a 3' LTR were found to generate intact HaSV at the nt, because NdN lacks the 3400 HaSV-specific nt between the titer of 104 focus-forming units per ml upon superinfection with nontransforming helper retrovirus. The intact HaSV was pre- 1 2 3 4 sumed to have resulted from recombination with helper virus (4). us - Origin However, it has been demonstrated by that HaSV generation in this system was dependent not on recombination with a nontransforming helper retrovirus but on the nontran- -23 All ccctacattg aaacatcagc caagacccgg cagggtgtag aggatgcctt -4.4 "______ctacacacta gtacgtgaga ttcggcagca taaactgcgg aaactgaacc cgcctgatga gagtggccct ggctgcatga gctgcaagt tgtgctgtcc 2.2 2.0 All but pFN 7 tgacaccagg tgaggcaggg accagcaaga catctggggc agtggcctca

pFN 7 .4 ...... 4 3.x.. 4x. xx.. 2.. All but pFN 7 gctagc/Nhe -0.6 pFN7 4. FIG. 6. The 3' terminal 206 nt of four NdN viruses, which lack the 3' ppt and the native 5' border of the LTR (Fig. 2), are compared to their equivalent from HaSV. The sequences of the four cloned NdN viral DNA fragments pFN1, -3, -5, and -7 (see text), generated by PCR, FIG. 5. ras/LTR recombination interface of NdN virus is colinear extend upstream from an Nhe I restriction site that defines position with that of the input NdN provirus construct. The ras/LTR interface 1698 in wild-type HaSV (1) and that defines the 5' border of the 3' of NdN provirus from DNA of NdN virus-transformed C3H10T½/2 LTR of the four NdN viruses. The sequences were derived by Lark cells was amplified by PCR with HaSV-specific primers. After ampli- Sequencing Technologies (Houston), as described previously. All, all fication, the DNA was coelectrophoresed through 1% agarose (lane four NdN sequences and the HaSV equivalent are identical. Specific 2) with DNA from clone 8 cells (lane 3) or NdN plasmid (lane 1) nucleotides were observed in pFN7; 4 is probably g, 3 is probably a, 2 amplified with the same primers. The sizes ofHindIII-digested A phage is probably t, x could be any nucleotide, and . means that the nucleotide DNA markers (lane 4) are identified on the right in kb. is the same as in the other FN fragments. Downloaded by guest on September 25, 2021 2464 Biochemistry: Schwartz et al. Proc. Natl. Acad Sci. USA 92 (1995) scribed promoter region of the 5' LTR of the 3'-truncated an earlier study by Sorge and Hughes (11), who reported that Harvey sarcoma provirus. Virus regeneration in this system a ppt deletion mutant failed to replicate upon transfection. was achieved by tandemization or concatenation of the input However, since the mutant did not include a selectable marker, DNA during transfection (3, 8). The resulting HaSVs probably such as a viral oncogene, it is not clear whether the failure of had 3' ras/LTR junctures exactly analogous to the NdN and Su the mutant to replicate was due to the deletion of the ppt or constructs described here-i.e., the 5' LTR of one Harvey to some other defect. Such a marker can be used to distinguish sarcoma provirus functioned as the 3' LTR of another after between an infectious DNA that produces a transforming two plasmids had been linked by illegitimate recombination RNA that is replication competent and one that is not (8). during transfection (3, 8). Our result that the 5' 30 nucleotides of the retroviral LTR Experiments by others designed to distinguish between the are not essential for replication confirms and extends earlier two models avoided self-recombination of input DNA during studies (8) but may be at variance with others. According to transfection and observed onlyvery low rates of recombination some investigators the 5'-most nucleotides of both LTRs with helper virus- e.g., titers of 0-100 recombinant virus units include a terminal complementarity that is thought to be per ml (5-7). The results of these experiments favor the DNA essential for provirus integration (2). model in the following ways: (i) Stuhlmann et al. (5) observed Further work is necessary to determine whether the 10-fold that recombination was "unexpectedly" independent of how higher titers of wild-type HaSV compared with our recombi- efficiently viral RNA that was transcribed from transfected nant viruses are due to the presence of a ppt. Nevertheless, proviral DNA constructs was packaged into virus particles- since the ppt-free transforming viruses studied here replicate exactly as predicted by the DNA model. (ii) Swain and Coffin to a high titer, they could acquire ppt by semilegitimate DNA (6) observed that parental viral sequences were inverted recombination with helper provirus. During this process re- during recombination. Sequence inversion is not observed combination would be initiated between a homologous region during reverse transcription (1) but it is consistent with of the common 3' LTRs and resolved at nonhomologous numerous precedents in DNA recombination. (iii) Zhang and regions 3' of the coding region of the protooncogene and 5' of Temin (7) reported nonhomologous RNA recombination to the ppt of the helper virus. occur at rates of "0.1 to 1 percent of the rate of homologous recombination." However, after 30 years of propagating Rous We thank Paul Berg, who has studied retroviral transduction by sarcoma virus (RSV) strains without envelope genes, an means of RNA recombination, for a critical review and constructive comments. This investigation was supported in part by the Council for envelope-positive RSV generated by recombination with en- Tobacco Research and private donations from Glenn Braswell (Los velope-positive helper viruses has never been observed (1, 18, Angeles), Dr. Richard Fischer (Annandale, VA), Dr. Fabio Franchi 19). This observation argues against any illegitimate recom- (Trieste, Italy), Dr. Friedrich Luft (Berlin), and Dr. Peter Paschen bination at detectable rates, in natural conditions of infection, (Hamburg). particularly because envelope-positive RSV strains exist naturally (1). Likewise, propagation of other defective transform- 1. Weiss, R., Teich, N., Varmus, H. & Coffin, J. (1985) Molecular ing viruses with corresponding helper viruses has never been Biology of RNA Tumor Viruses (Cold Spring Harbor Lab. Press, observed to generate nondefective transforming retroviruses Plainview, NY). (20). Thus, the relatively high rates of nonhomologous recom- 2. Brown, P. O., Bowerman, B., Varmus, H. E. & Bishop, J. M. (1987) Cell 49, 347-356. bination observed experimentally by Zhang and Temin may 3. Goodrich, D. W. & Duesberg, P. H. (1990) Proc. Natl. Acad. Sci. again have reflected recombination of unintegrated proviral USA 87, 3604-3608. DNAs during transfection. (iv) Three studies claim the pres- 4. Goldfarb, M. P. & Weinberg, R. A. (1981) J. Virol. 38, 125-135. ence of poly/oligo(A) tracts at the 3' junction of a transduced 5. Stuhlmann, H., Dieckmann, M. & Berg, P. (1990) J. Virol. 64, sequence as support of the RNA model. One claims 10 A 5783-5796. residues near the 3' end of a protooncogene sequence in a 6. Swain, A. & Coffin, J. M. (1992) Science 255, 841-845. natural sarcomagenic retrovirus (21). Two others claim either 7. Zhang, J. & Temin, H. M. (1993) Science 259, 234-238. internal oligo(A) tracts in 2 of about 50 (6) or in an undeter- 8. Goodrich, D. W. & Duesberg, P. H. (1988) Proc. Natl. Acad. Sci. USA 85, 3733-3737. mined number of in vitro recombinants (7) as evidence for 9. Olson, P., Temin, H. M. & Dornburg, R. (1992) J. Virol. 66, RNA recombination between the poly(A) tracts of protoon- 1336-1343. cogene mRNAs and retroviral RNAs. However, a recombi- 10. Varmus, H. E., Heasly, S., Kung, H.-J., Oppermann, V. C., nant virus with a cell-derived poly(A) sequence necessarily Smith, J. M., Bishop, J. M. & Shank,'P. R. (1978)J. Mol. Biol. 120, also includes the preceding cell-derived polyadenylylation 55-82. signal. Such a virus would not be stable, as it would prema- 11. Sorge, J. & Hughes, S. H. (1982) J. Virol. 43, 482-488. turely terminate viral RNA and thus eliminate the essential 12. Khan, A. 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(1972) Nature (London) 235, functions like efficient illegitimate recombination (22). 383-386. Neither a ppt nor the 5' 30 Nucleotides of the LTR Appear 18. Hanafusa, H., Hanafusa, T. & Rubin, H. (1963) Proc. Natl. Acad. Sci. USA 49, 572-580. Essential for Retrovirus Replication. The relatively high titers 19. Weiss, R. A., Mason, W. S. & Vogt, P. K. (1973) Virology 52, of 105 focus-forming units per ml of Su virus and NdN virus 535-552. show that neither ppt nor even the 5'-most 30 nucleotides of 20. Maisel, J., Dina, D. & Duesberg, P. (1977) Virology 76, 295-312. the LTR are essential for virus replication. The result that the 21. Huang, C. C., Hay, N. & Bishop, J. M. (1986) Cell 44, 935-940. ppt sequence is not essential for replication is at variance with 22. Weiss, R. A. (1994) Science 264, 1954-1955. Downloaded by guest on September 25, 2021