INTRODUCTION

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Retroviruses package two copies of viral RNA into each virion (9, 28). During reverse transcription, both copackaged RNAs can be used as templates to produce a recombinant with a mixture of genetic information from each of the parental RNAs (8, 18). Retroviruses recombine at high rates (6, 15, 23, 24, 26, 29, 30, 46-48). Using murine leukemia virus (MLV)-based vectors with markers separated by 1.0, 1.9, and 7.1 kb, recombination rates of 4.7, 7.4, and 8.2%, respectively, were observed in one round of viral replication (2).

During reverse transcription of the viral genome, the virus-encoded enzyme reverse transcriptase (RT) has to perform two template-switching events (named minus- and plus-strand DNA transfer) to complete the synthesis of viral DNA (12). It has been hypothesized that RT is evolutionarily selected to have a low affinity to the template and low processivity in order to complete the two obligatory template-switching events (44). A consequence of this low processivity is that RT may also perform other nonobligatory template-switching events during reverse transcription. Because two copies of RNA are present in the virion, after dissociation from the template used for DNA synthesis, RT may reassociate with the same RNA or switch to the copackaged RNA, resulting in intramolecular or intermolecular template-switching events, respectively. Between these two events, only intermolecular template switching generates recombination.

The rates of intramolecular and intermolecular template switching were measured in a spleen necrosis virus (SNV)-based system (17). It was found that intramolecular template switching occurred far more frequently than intermolecular template switching. Furthermore, when recombination was selected in one region of the viral genome, intermolecular template switching was more likely to be observed in another region of the genome. In contrast, when recombination was not selected, intermolecular template switching was not observed in a different region of the viral genome; however, frequent intramolecular template switching was still observed. This observation led to the hypothesis that two viral populations exist. In the first population (recombining population), RT could frequently switch templates to the same RNA or the copackaged RNA. In the second population (nonrecombining population), RT could only switch to the same RNA frequently. Because frequent template switching occurred in both populations, the difference seemed to be the ability of the RT to interact with the other template. We hypothesized that if the barrier to intermolecular template switching is the ability to access the other RNA, then it should be possible to increase recombination by bringing the two copackaged RNAs closer together. In our experimental system, recombination is measured by the presence of two selectable markers, one from each parent, in the progeny provirus. Therefore, to test our hypothesis, the region between the two selectable markers from the two copackaged RNAs should be in close proximity to observe any effect on recombination.

Electron microscopy studies demonstrated that the two copackaged retroviral RNAs were dimerized at the 5' end of the RNA (27, 28, 32, 39). Genetic and biochemical studies defined a region important for dimerization through noncovalent interaction between the two viral RNAs (13, 40, 42). The region important for dimerization of MLV RNAs, the dimer linkage structure (DLS), is part of the packaging signal () located within the 5' untranslated region (UTR) (11, 13, 39, 40, 42). In the current report, we examined the effect of the naturally occurring dimerization between the two copackaged RNAs on recombination. The extended MLV packaging signal (⁺), including the DLS, was relocated from the 5' UTR to the middle of the viral genome between two selectable markers. Recombination rates between vectors with ⁺ located in the 5' UTR were directly compared to those from vectors with ⁺ located between the selectable markers.

In addition, using a forced-recombination experiment, it was previously observed that the DLS was a putative recombination hot spot (31, 34, 35). To determine the general effect of the DLS on the location of template-switching events, a set of vectors that contained five sets of restriction enzyme markers was used to dissect the locations of template-switching events after one round of retroviral replication.

	MATERIALS AND METHODS

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Plasmid construction. Vectors JS30 and JA32-1kb have been described previously (2); to avoid confusion, JA32-1kb is abbreviated as JA32 in this report. Vectors pJA33-1.3kb, pJA9, pJA10Neo, pJA11Hy, pJA23, pJA19Neo, and pJA20Hy were constructed by standard molecular cloning techniques (33). All of the MLV-based vectors were constructed with various derivatives of pLAEN (38) as backbones. To generate pJA33-1.3kb, pJS30 (2) was partially digested with NdeI, treated with calf intestinal phosphatase (CIP), and ligated to a linker (5'-TAACGCGT-3') to create a unique MluI site and an 8-bp insertion in the hygromycin phosphotransferase B gene (hygro) (14). To generate pJA9, pWH390-Cla was digested with EcoRI and ligated to annealed linkers PL1 (5'-AATTAGGCCTCATATGCTAGCCTCGAG-3') and PL2 (5'-AATTCTCGAGGCTAGCATATGAGGCCT-3') to generate pBWB-1. pBWB-1 was digested with ClaI and treated with CIP and Escherichia coli DNA polymerase I large fragment (Klenow) to fill in the recessive ends. This treated pBWB-1 DNA was ligated to a 1.0-kb DNA fragment containing hygro to generate pJA30BWB2. pJA30BWB2 was digested with XhoI plus BclI, treated with Klenow fragment to fill in the recessive ends, and self-ligated to remove the 0.6-kb sequence containing the internal ribosomal entry site (IRES) (1, 19, 20) to form pCN2. pCN2 was digested with XhoI, treated with CIP, and ligated to annealed linkers 5' neoJA (5'-TCGAGGTCGACGCGGCCGCCA-3') and 5'oenJA (5'-TCGATGCGGCCGCGTCGACC-3'). The resulting plasmid, pJA5, contained unique XhoI and NotI restriction sites immediately upstream of the neomycin phosphotransferase gene (neo) (22). pJA5 was partially digested with BamHI, treated with Klenow fragment to fill in recessive ends, and self-ligated to generate pJA6, which contains a unique BamHI site immediately downstream of hygro. pJA6 was digested to completion with BamHI, treated with CIP, and ligated to annealed linkers 3'hygroJA (5'-GATCCGTCGACAAGCTT-3') and 3' orgyhJA (5'-GATCAAGCTTGTCGACG-3'). The resulting plasmid, pJA7, contained unique BamHI and HindIII restriction sites immediately downstream of hygro. pJA7 was digested with BamHI and HindIII, treated with CIP, and ligated to annealed linkers Jarzts1 (5'-GATCCGAATGCATCGTGTCAAGTTAGGTCTGCGTA-3') and JA1stzr (5'-AGCTTACGCAGACCTAACTTGACACGATGCATTCG-3') to generate pJA8. pJA8 was digested with XhoI plus NotI, treated with CIP, and ligated to annealed linkers Jats2 (5'-TCGAGCATGCATATGTCTCTAACTCTTCGATGCCCGTTCTGTCTACTTGTGC-3') and JA2st (5'-GGCCGCA CAAGTAGACAGAACGGGCATCGAAGAGTTAGAGACATATGCATGC- 3') to generate pJA9. To generate pJA10Neo, pJA9 was partially digested with SacII, treated with T4 DNA polymerase to remove the protruding 2 bp at the 3' termini, and self-ligated. The resulting plasmid, pJA10Neo, contained an NaeI site in the inactivated hygro. To generate pJA11Hy, pJA9 was partially digested with NarI (an isoschizomer of EheI), treated with Klenow fragment to fill in recessive ends, and self-ligated. These procedures introduced a BssHII site and inactivated neo in pJA11Hy. pJA9 was partially digested with FspI, treated with CIP, and ligated to a linker (5'-GGACGTCC-3'); these procedures generated pJA14, which contained an 8-bp insertion and an AatII site in neo. pJA15 was generated by partial digestion of pJA9 with NcoI followed by Klenow fill-in reaction and self-ligation. These procedures introduced a 4-bp insertion to generate an NsiI site and inactivated hygro. pJA16 was generated by complete digestion of pJA14 with BamHI followed by Klenow fill-in reaction and self-ligation; these procedures introduced a ClaI site immediately downstream of hygro. pJA17 was generated by complete digestion of pJA15 with XhoI, followed by Klenow fill-in reaction and self-ligation; these procedures generated a PvuI site immediately upstream of neo. pJA17 was partially digested with AflII, treated with CIP, and ligated to a linker, JAmlu (5'-TTAACGCG-3'), to generate pJA18; these procedures introduced a unique MluI site in the gag-coding region included in the MLV extended packaging signal ⁺. To generate pJA19Neo, the splice donor site of pJA18 was mutated from AGGTAAG to TCGACAG by overlapping PCR mutagenesis that also created a unique SalI site. To generate pJA20Hy, pJA16 was digested with AscI plus EcoRI, treated with CIP, and ligated to the 0.76-kb AscI-EcoRI-digested DNA fragment from pJA19Neo. pJA23 was constructed by digesting pJA9 with AscI plus EcoRI, treating it with CIP, and ligating it to the same 0.76-kb DNA fragment from pJA19Neo. All of the plasmids were characterized by restriction enzyme mapping to confirm the general structures. DNA sequencing was performed to ensure that the PCR-amplified DNA used for cloning did not introduce any inadvertent mutations. In addition, DNA sequencing was also performed in some plasmids to ensure that only one linker was inserted into the plasmids.

Cells, DNA transfections, and virus propagations. All cells were obtained from the American Type Culture Collection. PA317 is a murine cell line that expresses MLV gag-pol and env (36). PG13 is a murine cell line that expresses MLV gag-pol and gibbon ape leukemia virus (GaLV) env (37). D17 is a dog osteosarcoma cell line permissive to infection by MLV (41).

Southern hybridization analysis. Genomic DNA purification, digestion, and hybridization were performed by standard molecular techniques (33). DNA transfers were performed with a vacuum blotter (Pharmacia). All blots were hybridized with probe generated from either a 1.3-kb MluI-EheI fragment of pJA33-1.3kb, a 1.9-kb NcoI-NcoI fragment of pJS30, or a 1.3-kb NcoI-FspI fragment of pJS30. Probes were generated by the random-priming method with [-³²P]dCTP (specific activity of >10⁹ cpm/µg of DNA) (10). Southern hybridization results were obtained by autoradiography or PhosphorImager analysis (Molecular Dynamics).

Mapping of recombinant proviruses. DNA lysates were prepared from double-drug-resistant cell clones and used as substrates for PCR (16). Proviral genomes were amplified with hygro- and neo-specific primers JA16-995 (5'-GGATATGTCCTGCGGGTAAATAGC-3') and 16AJ-3327 (5'-ATCGACAAGACCGGCTTCCATCCG-3'), respectively. All PCRs were carried out in a Hybaid Omnigene thermal cycler for 35 cycles. Amplified DNAs were analyzed with various restriction enzyme digestions, separated by gel electrophoresis, and visualized by ethidium bromide staining. All DNA manipulations were performed by standard procedures (33).

	RESULTS

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Retroviral vectors used to study the effect of the MLV extended packaging signal on retroviral recombination. To examine the effect of the MLV ⁺ on retroviral recombination, we constructed a set of vectors in which ⁺ was located in the 5' UTR and another set of vectors in which ⁺ was located in the middle of the viral genome between two selectable markers. Both sets of vectors were derived from MLV and contained cis-acting elements required for viral replication and gene expression. The first set of vectors (pJS30, pJA33-1.3kb, and pJA32) contained ⁺ in the 5' UTR (Fig. 1). These vectors also contained hygro and neo; both genes were expressed by transcripts initiated from the long terminal repeat (LTR). The translation of neo was directed by an IRES from encephalomyocarditis virus (1, 19, 20). These three vectors were highly homologous (>99%) to one another. Vector pJS30 contained functional hygro and neo (14, 22). Vector pJA33-1.3kb contained a functional neo and a nonfunctional hygro with an 8-bp frameshift insertion that destroyed an NdeI site and generated an MluI site. Vector pJA32 (2) contained a functional hygro and a nonfunctional neo with a 4-bp frameshift insertion that destroyed an EheI site and generated a BssHII site. The distance between the two inactivating mutations in hygro and neo was 1.3 kb. Frameshift mutations of more than 1 bp were used to inactivate genes because they exhibit a low reversion rate (2, 18).

View larger version (39K): [in this window] [in a new window]   FIG. 1.   MLV vectors used to determine the role of the extended packaging signal (+) in retroviral recombination. sd, mutated splice donor site; *, inactivating frameshift mutation; Nd, NdeI; E, EheI; M, MluI; Bs, BssHII; S, SacII; Na, NaeI; Ns, NsiI; B, BamHI; P, PvuI; F, FspI; Nc, NcoI; C, ClaI; Af, AflII; X, XhoI; Aa, AatII. Translation of Neo in these constructs was directed by IRES from encephalomyocarditis virus or from MLV +.

Experimental protocol used to measure the rates of recombination in one replication cycle. The protocol used to determine the rate of homologous recombination is illustrated in Fig. 2 with vector set pJA33-1.3kb and pJA32 as an example. Vectors pJA33-1.3kb and pJA32 were separately transfected into PA317 helper cells, selected with either G418 or hygromycin, and the resulting colonies were pooled separately. The pool size for each vector was greater than 160 colonies. Viruses were harvested from these two PA317 cell pools and used to infect PG13 helper cells simultaneously. PG13 cell clones resistant to hygromycin plus G418 were isolated, and the proviral structures in these cell clones were characterized by Southern hybridization analysis. Cell clones containing one intact copy of each of the JA33-1.3kb and JA32 proviruses were selected. Three types of virions can be produced from these dual-infected PG13 cells: virions containing two copies of JA33-1.3kb RNA, virions containing two copies of JA32 RNA, and virions containing one copy of each of the JA33-1.3kb and JA32 RNAs (heterozygotic virions). The first two types of virions generate proviruses containing one functional drug resistance gene that confers resistance to a single drug. Recombination can occur in the heterozygotic virions to generate proviruses that contain two functional drug resistance genes that confer resistance to both drugs (18, 48). Viruses were harvested from the selected PG13 cell clones and used to infect D17 target cells. Infected D17 cells were subjected to single (hygromycin or G418) or double (hygromycin plus G418) drug selection. Virus titers were then determined by counting the numbers of drug-resistant colonies. Recombination rates were calculated from the double- and the single-drug-resistant colony titers (18). In addition, double-drug-resistant D17 cell clones were isolated, and Southern hybridization analysis was performed to confirm the recombinant genotype of the proviruses.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Experimental protocol to measure the rates of recombination. Abbreviations and symbols are defined in the legend to Fig. 1.

The control vectors generate similar single- and double-drug-resistant colony titers. In this system, recombination rates were calculated from single- and double-drug-resistant titers. Therefore, it was important to determine whether the three different drug treatments generated similar viral titers in infected cells.

Characterization of proviral structures in PG13 cell clones infected with JA33-1.3kb and JA32. PG13 cell clones containing JA33-1.3kb and JA32 were generated to measure the recombination rate between markers separated by 1.3 kb with ⁺ located in the 5' UTR. The proviral structures in these cell clones were characterized by Southern hybridization analysis. Partial restriction enzyme maps of JS30, JA33-1.3kb, and JA32 are shown in Fig. 3A. The JS30 provirus had a unique NdeI site located in hygro and four EheI sites (one in each LTR, one in ⁺, and one in neo). In JA33-1.3kb, the NdeI site was destroyed to inactivate hygro, whereas in JA32, an EheI site was destroyed to inactivate neo. All three proviruses contained four EcoRV sites, two in each LTR. When genomic DNA from cell clones containing a copy of JA33-1.3kb and a copy of JA32 was digested with EheI, NdeI, and EcoRV and hybridized with probes generated from a 1.3-kb MluI-EheI fragment of JA33-1.3kb, a 1.8- and a 2.4-kb band were expected from the JA33-1.3kb and the JA32 proviruses, respectively (Fig. 3A). A representative Southern blot of three different PG13 cell clones (A2, B1, and E1) is shown in Fig. 3B; each cell clone contained the expected bands. A unique HindIII site was located in JA33-1.3kb and JA32 proviruses at the 5' end of IRES (Fig. 3A). Therefore, each provirus was expected to generate two bands when digested with HindIII and hybridized with the aforementioned probe. Since retroviruses integrate randomly into the host genome (7), the band sizes should vary according to the site of integration. Therefore, cell clones that contain one copy of JA33-1.3kb and JA32 should produce four bands of different sizes upon HindIII digestion and Southern analysis. A representative Southern analysis with HindIII digestion of genomic DNA from the helper cell clones is shown in Fig. 3B; all cell clones (Fig. 3B and data not shown) exhibited a different band pattern, indicating that they were derived from independent infection events (Fig. 3B, lanes H).

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Proviral structures of JA33-1.3kb, JA32, and JS30. Recombinants with two functional drug resistance genes have the same structure as JS30. (A) Predicted proviral structures. RV, EcoRV; H, HindIII; zigzag lines, host cell sequences. A 1.3-kb MluI-EheI DNA fragment of JA33-1.3kb was used for a random-priming reaction to generate a probe for Southern hybridization analysis; the asterisk in JA33-1.3kb denotes the MluI site. This probe hybridized to the 3' end of hygro and the 5' end of neo and is indicated by the black box. (B) Southern hybridization analysis of the proviral structures in virus-producing and target cell clones. A2, B1, and E1 are PG13 virus-producing cell clones containing a copy of each of the JA33-1.3kb and JA32 proviruses. A2-1a, B1-b, and E1-b are double-drug-resistant D17 cell clones infected with viruses harvested from A2, B1, and E1, respectively. Other abbreviations and symbols are defined in the legend to Fig. 1.

Recombination rate of MLV vectors with ⁺ located in the 5' UTR and markers separated by 1.3 kb. Viruses harvested from eight independent PG13 cell clones that contained a copy of JA33-1.3kb and JA32 were used to infect D17 target cells. Viral titers are shown in Table 2. The hygromycin-resistant colony titers ranged from 0.15 × 10⁵ to 33.0 × 10⁵ CFU/ml, the G418-resistant colony titers ranged from 0.18 × 10⁵ to 31 × 10⁵ CFU/ml, and the hygromycin-plus-G418-resistant colony titers ranged from 0.022 × 10⁴ to 8.9 × 10⁴ CFU/ml.

Characterization of proviral DNA structures in PG13 cells infected with JA10Neo and JA11Hy. PG13 cell clones containing JA10Neo and JA11Hy were generated to examine the effect of ⁺ located between the selectable markers on the rate of recombination. Southern analyses were performed to examine the proviral structures in coinfected PG13 cell clones. Partial restriction enzyme maps of JA9, JA10Neo, and JA11Hy are shown in Fig. 4A. JA9, JA10Neo, and JA11Hy proviruses each contained a unique ClaI site between the 5' LTR and hygro. JA9 proviruses contained four EheI sites: one in each LTR, one in ⁺, and one in neo. JA10Neo proviruses contained all four EheI sites, whereas JA11Hy contained only three EheI sites, because one of the sites was mutated to inactivate neo. A unique SacII site was located in hygro of JA9 and JA11Hy; this site was mutated in JA10Neo to inactivate hygro. Genomic DNAs from PG13 cell clones coinfected with JA10Neo and JA11Hy were digested with three enzymes: EheI, SacII, and ClaI. Five bands were expected from this digestion when hybridized with a probe containing the 3' portion of hygro and 5' portion of neo. A 1.4-kb band and a 1.9-kb band were expected to be generated from the JA10Neo provirus, whereas 0.8-, 1.1-, and 1.6-kb bands were expected to be generated from the JA11Hy provirus (Fig. 4A). A representative Southern blot of three different PG13 cell clones (C1, C2, and A4) is shown in Fig. 4B. Each cell clone contained the expected fragments consistent with the predicted structures of JA10Neo and JA11Hy. In addition, all DNAs were digested with HindIII to confirm that all of the PG13 cell clones used were generated through independent infection events (Fig. 4B and data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Proviral structures of JA10Neo, JA11Hy, and JA9. Recombinants with two functional drug resistance genes have the same structure as JA9. (A) Predicted proviral structures. A 1.9-kb NcoI-NcoI DNA fragment from JS30 was used for a random-priming reaction to generate a probe for Southern hybridization analysis. This probe hybridized to the 3' end of hygro and the 5' end of neo and is indicated by the black boxes. (B) Southern hybridization analysis of the proviral structures in virus-producing and target cell clones. C1, C2, and A4 are PG13 virus-producing cell clones infected with JA10Neo and JA11Hy. C1-b, C2-b, and A4-b are double-drug-resistant D17 cell clones infected with viruses harvested from C1, C2, and A4, respectively. Other abbreviations and symbols are defined in the legends to Fig. 1 and 3.

Recombination rate of MLV vectors with ⁺ located between two selectable markers that were 1.3 kb apart. Six PG13 cell clones coinfected with JA10Neo and JA11Hy were identified; viruses were harvested from these cell clones and were used to infect D17 target cells. The infected target cells were placed on hygromycin, G418, or hygromycin-plus-G418 drug selection. Virus titers generated from these six cell clones are shown in Table 3. The hygromycin-resistant colony titers ranged from 1.1 × 10⁵ to 5.4 × 10⁵ CFU/ml, the G418-resistant colony titers ranged from 0.50 × 10⁵ to 1.9 × 10⁵ CFU/ml, and the hygromycin-plus-G418-resistant colony titers ranged from 3.1 × 10³ to 13.4 × 10³ CFU/ml.

View this table: [in this window] [in a new window]   TABLE 3.   Virus titers generated from PG13 cells coinfected with JA10Neo and JA11Hy

Effect of ⁺ on the rate of recombination when markers were separated by 1.3 kb. The recombination rate of vectors with markers separated by 1.3 kb when ⁺ was located in the 5' UTR or between selectable markers was 5.0 or 12.0%, respectively. These data indicated that the recombination rate was approximately twofold higher when ⁺ was located between the selectable markers. This increase is statistically significant (P = 0.00002; one-way analysis of variance [ANOVA]). This higher rate of recombination could be caused by an increase in the size of viral population that had the potential to undergo recombination (recombining population). Alternatively, the size of the recombining population might not have changed; however, a larger proportion of the viruses within the recombining population might have undergone recombination between the selectable markers when ⁺ was relocated to the middle of the genome. We previously observed that when ⁺ was located in the 5' UTR, recombination rates increased linearly between marker distances of 1.0 kb (4.7%) and 1.9 kb (7.4%) (2). If the recombining population had been altered, we should observe an increase in the recombination rate when the marker distance was increased. However, if the recombining population had not changed, we should not observe an increase in recombination rates when the marker distance was increased because the observed rate was already close to the maximum rate detected (8.2%) (2). To distinguish between these two possibilities, we measured recombination rates between markers 1.9 kb apart.

Vectors used to further study the effect of ⁺ on the rate and location of the recombination events. To further characterize the effect of ⁺ on recombination, a third set of vectors was constructed to measure the recombination rate when markers were separated by 1.9 kb. The vectors pJA23, pJA19Neo, and pJA20Hy were very similar in sequence to the second set of vectors, pJA9, pJA10Neo, and pJA11Hy, respectively. The structures of these viral vectors are shown in Fig. 1.

Proviral DNA analysis of PG13 cell clones infected with JA19Neo and JA20Hy. PG13 cell clones containing JA19Neo and JA20Hy were generated to further examine the effect of ⁺ on recombination. Southern hybridization analyses were performed to determine the proviral structures. Partial restriction enzyme maps of JA23, JA19Neo, and JA20Hy are shown in Fig. 5A. JA23, which has functional hygro and neo, has an NcoI site located in each drug resistance gene and a unique FspI site located in neo. One of the NcoI sites was destroyed to inactivate hygro in JA19Neo, whereas the FspI site was destroyed to inactivate neo in JA20Hy. In addition, all three vectors contained an XbaI site located in each LTR.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Proviral structures of JA19Neo, JA20Hy, and JA23. Recombinants with two functional drug resistance genes have the same structure as JA23. (A) Predicted proviral structures. Xb, XbaI. A 1.3-kb NcoI-FspI fragment of JS30 was used for a random-priming reaction to generate a probe for Southern hybridization analysis. This probe hybridized to the 3' end of hygro and the 5' end of neo and is indicated by the black boxes. (B) Southern hybridization analysis of the proviral structures in virus-producing and target cell clones. Clones 1, 7, and 15 are PG13 virus-producing cell clones infected with JA19Neo and JA20Hy. 1-2c, 7-8a, and 15-14b are double-drug-resistant D17 cell clones infected with viruses harvested from clones 1, 7, and 15, respectively. Other abbreviations and symbols are defined in the legends to Fig. 1 and 3.

Recombination rate of MLV vectors with ⁺ located between selectable markers that were 1.9 kb apart. Viruses were harvested from five independent PG13 cell clones that contained a copy each of JA19Neo and JA20Hy and used to infect D17 target cells. Viral titers are shown in Table 4. The hygromycin-resistant colony titers ranged from 0.50 × 10⁵ to 7.8 × 10⁵ CFU/ml, the G418-resistant colony titers ranged from 0.20 × 10⁵ to 20 × 10⁵ CFU/ml, and the hygromycin-plus-G418-resistant colony titers ranged from 1.2 × 10³ to 5.6 × 10³ CFU/ml.

View this table: [in this window] [in a new window]   TABLE 4.   Virus titers generated from PG13 cells coinfected with JA19Neo and JA20Hy

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Strategy for mapping recombinant proviruses in cell clones. (A) Structures of the internal portions of the parental viruses. JA19Neo is shown in black, and JA20Hy is shown in white. Restriction enzyme sites are indicated above or below each parental genotype. The two selectable markers are indicated by the open circles. Arrows, hygro- and neo-specific primers for PCR; other abbreviations and symbols are the same as in Fig. 1. (B) Restriction enzyme maps of 42 recombinant proviruses containing one or three template switches. JA19Neo-derived sequences are shown in black, whereas JA20Hy-derived sequences are shown in white. The number to the right of each genotype indicates the number of recombinants with the same genotype observed in the 42 proviruses analyzed. (C) Summary of template-switching events. Restriction enzyme markers corresponding to JA19Neo and JA20Hy are shown above and below a generic recombinant structure, respectively. The distances between each set of restriction enzyme markers for four different regions are indicated below recombinant structure. In addition, the expected and observed frequencies of template-switching events are shown beneath the four regions. Expected frequencies were calculated by dividing the distance between each set of markers by the total distance between the selectable markers. Abbreviations are the same as those in Fig. 1 and 6.

PCR amplification and restriction enzyme analysis of recombinant proviruses generated from JA19Neo and JA20Hy. The molecular natures of the recombinant proviruses generated from JA19Neo and JA20Hy were examined. To ensure that independent recombination events were studied, most of the cell clones were isolated from different cell culture dishes. For cell clones isolated from the same culture dishes, Southern hybridization analyses were performed to ensure that these proviruses had arisen through independent infection events (data not shown). A portion of the proviral genome (2.3 kb) was amplified by PCR with primers specific to hygro and neo (Fig. 6A). Restriction enzyme mapping was performed on PCR products to determine the molecular nature of the recombinant proviruses. For each DNA sample, seven different single-enzyme digestions (NsiI, BamHI, ClaI, MluI, AflII, PvuI, and XhoI) and two double-enzyme digestions (AatII plus SacII and NcoI plus FspI) were performed. The genomes of 42 recombinant proviruses were analyzed and are shown in Fig. 6B. Of the 42 recombinant proviruses analyzed, 34 contained one template switch and 8 contained three template switches in the 2.3-kb region amplified by PCR. Among the 34 proviruses with one template switch, recombination occurred in all four regions between the five sets of restriction enzyme markers. Of these template-switching events, 8 occurred in the 5' portion of neo between FspI of JA19Neo and XhoI of JA20Hy, 1 occurred in the 3' portion of ⁺ between PvuI of JA19Neo and AflII of JA20Hy, 12 occurred in the 5' portion of ⁺ between MluI of JA19Neo and ClaI of JA20Hy, and 13 occurred in the 3' portion of hygro between BamHI of JA19Neo and NcoI of JA20Hy (Fig. 6B). Of the proviruses containing three template switches, four patterns were observed. Three proviruses had the first template switch between PvuI of JA19Neo and AflII of JA20Hy, the second template switch between AflII of JA20Hy and BamHI of JA19Neo, and the third template switch between BamHI of JA19Neo and NcoI of JA20Hy. Two proviruses had the first template switch between FspI of JA19Neo and XhoI of JA20Hy, the second template switch between XhoI of JA20Hy and MluI of JA19Neo, and the third template switch between BamHI of JA19Neo and NcoI of JA20Hy. Two proviruses had the first template switch between FspI of JA19Neo and XhoI of JA20Hy, the second template switch between AflII of JA20Hy and BamHI of JA19Neo, and the third template switch between BamHI of JA19Neo and NcoI of JA20Hy. One provirus had the first template switch between FspI of JA19Neo and XhoI of JA20Hy, the second template switch between XhoI of JA20Hy and MluI of JA19Neo, and the third template switch between MluI of JA19Neo and ClaI of JA20Hy.

Distribution of the template-switching events in the recombinant proviruses. During reverse transcription, recombination had to occur to obtain the NcoI site in hygro and the FspI site in neo to generate recombinants with functional hygro and neo. In the region between these two sets of selectable markers, JA19Neo and JA20Hy differed in three other sets of restriction enzyme sites. These five sets of markers divided the 1.9-kb region between the selected markers into four regions (Fig. 6C). Region 4 was located between NsiI-NcoI and BamHI-ClaI markers; this region was 0.66 kb in length and contained the 3' portion of hygro. Region 3 was located between the BamHI-ClaI and MluI-AflII markers; this region was 0.47 kb in length and contained the 5' portion of ⁺, including the DLS. Region 2 was located between the MluI-AflII and PvuI-XhoI markers; this region was 0.43 kb in length and contained the 3' region of the ⁺. Region 1 was located between the PvuI-XhoI and FspI-AatII markers; this region was 0.32 kb in length and contained the 5' portion of neo.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Recombination during retroviral replication provides an additional mechanism besides mutation to increase variation in viral populations. Although frequent recombination has been observed in most retroviruses (6, 15, 23, 24, 26, 29, 30, 46-48), many aspects of this phenomenon are still unclear. In this report, we tested whether the rate of recombination could be altered and determined the nature of this potential alteration. Specifically, we examined the effect of the extended packaging signal in recombination and determined the general effect of the DLS on the location of the template-switching events.

Recombination rates of MLV vectors with ⁺ located in the 5' UTR. Previously, we determined that in one round of replication, the homologous recombination rates with markers separated by 1.0, 1.9, and 7.1 kb were 4.7, 7.4, and 8.2%, respectively (2). In all of these vectors, ⁺ was located in the 5' UTR. It was found that the recombination rates reached a plateau when markers were separated by 1.9 kb and did not increase significantly even when the markers were further apart (7.1 kb). However, recombination rates increased as the distance between markers increased from 1.0 to 1.9 kb (4.7 and 7.4%, respectively). These data suggested that recombination rates increased in linear proportion when the distances between markers ranged from 1.0 to 1.9 kb. However, this relationship could not be determined because only two data points were obtained in the previous study. In the current study, a recombination rate of 5.0% ± 0.5% (SE) was determined with markers 1.3 kb apart. This rate is within the expected range calculated from the previous measured recombination rates when markers were separated by 1.0 and 1.9 kb (4.7%1.0 kb × 1.3 kb = 6.1%; 7.4%1.9 kb × 1.3 kb = 5.1%). Together with the previous published recombination rates, these data suggested that when the distances between the selectable markers were in the range of 1.0 to 1.9 kb, recombination rates increased linearly in proportion to the distance between markers. This is the first set of evidence to support that within a limited range, a linear relationship exists between recombination rates and the distances between markers.

The effect of the locations of ⁺ on the recombination rates of MLV vectors. It was previously observed that the entire viral population was capable of undergoing frequent template switching (17). However, only a subpopulation of viruses underwent recombination (intermolecular template switching). We hypothesized that the barrier to intermolecular template switching is the accessibility of the copackaged RNA. We suggested that the recombination rate may be altered by bringing the two copackaged RNAs closer together between the selectable markers by using the dimerization signal in ⁺. However, it was not clear whether the distance between the two copackaged RNAs was the only factor separating the viral subpopulation that could undergo recombination from the rest of the viruses. If the distance between the copackaged RNAs was the only factor, then the size of the subpopulation would be changed by relocating ⁺. In contrast, if the distance between the two RNAs was not the only factor, then it was quite possible that the size of the recombining subpopulation would not be drastically altered. To test our hypothesis, the recombination rates of MLV vectors with ⁺ located in the 5' UTR or between the selectable markers were compared. When selectable markers were separated by 1.3 kb, vectors with ⁺ located between hygro and neo had an approximately twofold-higher recombination rate than those from vectors with ⁺ located in the 5' UTR. This enhancement of recombination was less pronounced when the selectable markers were separated by 1.9 kb. The recombination rates of vectors with selectable markers separated by 1.3 and 1.9 kb were not significantly different when ⁺ was located between hygro and neo (12.0 and 10.4%, respectively; P = 0.30; one-way ANOVA). These data also suggested that the rate of recombination reached a plateau when the markers were separated by 1.3 kb. This was in sharp contrast with the observation that when ⁺ was located in the 5' UTR and markers were 1.0 to 1.9 kb apart, recombination rates were in linear proportion to the distances between the selectable markers.

Recombination in MLV exhibits high negative interference. High negative interference described a phenomenon in which a greater-than-expected probability of multiple recombination events was observed (3, 5, 17, 49). Using the observed recombination rate of 10.4% with ⁺ between markers separated by 1.9 kb, it was estimated that one in five proviruses should contain a single recombination event (see reference 17 for a detailed calculation). Since all of the proviruses analyzed in these experiments were recombinants, all proviruses contained at least one recombination event. Therefore, 1 in 5 and 1 in 25 recombinants would be expected to contain two and three recombination events, respectively. It was expected that recombinants with two template switches between the 1.9-kb region would contain a parental genotype and would not survive double-drug selection for functional hygro and neo. However, recombinants with three template-switching events could contain functional hygro and neo and should be observed. Analysis of 42 recombinant proviruses indicated that 34 contained one template switch, whereas 8 contained three template switches (Fig. 6B). Approximately 19% of the recombinant proviruses contained three template switches; this was fivefold greater than expected. Therefore, similar to the observations made with SNV (3, 17), homologous recombination in MLV also exhibited high negative interference. In addition, recombinants with multiple template switches were also frequently observed in human immunodeficiency virus type 1 (6, 50). Taken together, we propose that high negative interference is an inherent property of retroviral recombination.

The general effect of packaging signal on the locations of the template-switching events. Using a system selecting recombination in the MLV packaging signal, it was shown that template-switching events occurred frequently within a 33-nucleotide region that coincided with the DLS (31, 34, 35). These data suggested that DLS is a hot spot for recombination.