Department of Molecular and Structural Biology1 and Department of Medical Microbiology and Immunology,2 University of Aarhus, C. F. Moellers Allé, Bldg 130, DK-8000 Aarhus, Denmark
Author for correspondence: Finn Pedersen.Fax +45 86196500. e-mail fsp{at}mbio.aau.dk
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Abstract |
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Introduction |
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Encapsidation of two genomic RNA molecules into budding retrovirus particles allows for recombination during reverse transcription within the viral core particle internalized in the cytoplasm of the newly infected cell. Retrovirus recombination occurs primarily by template switching of nascent minus-strand DNA (Anderson et al., 1998 ; Coffin, 1979
; Hu & Temin, 1990a
, b
, 1992
; Hu et al., 1997
; Stuhlmann & Berg, 1992
), but may involve also events of plus-strand recombination (Junghans et al., 1982
; Mikkelsen et al., 1998a
). Coexistence and occasional copackaging of retroviral RNAs of exogenous and endogenous origin allow for the generation of recombinant proviruses harbouring sequences of both parental origins. In such a scenario, events of template switching during DNA synthesis may facilitate recombinational patch repair of virus mutations by substituting defective segments of the genome with functional sequence patches provided by the copackaged endogenous virus. Previously reported examples of ERV-based recombinational reversion include patch repair of virus mutants harbouring (i) modifications of the integrase attachment site (Colicelli & Goff, 1987a
, b
; Murphy & Goff, 1994
); (ii) deletions within IN and PR regions of the pol gene (Martinelli & Goff, 1990
; Schwartzberg et al., 1985
); and (iii) defective primer binding sites (PBS; Mikkelsen et al., 1996
, 1998b
). In addition, recombination events including both packaging construct and ERV-derived RNA have been found to result in `patch repair' of retrovirus vectors, leading to the hazardous generation of replication-competent viruses in retrovirus- based gene transfer applications (Chong et al., 1998
; Chong & Vile, 1996
; Vanin et al., 1994
).
RNA dimerization and encapsidation are tightly coupled events of retrovirus replication. Whereas copackaging of retroviral RNAs is a proven prerequisite for recombination (Hu & Temin, 1990a ), there remains an open question of whether RNA dimer formation is required for packaging and/or genetic interactions between copackaged RNAs. For all retroviruses studied, the primary determinants of RNA dimerization and encapsidation have been mapped to the packaging signal (
) within the 5' untranslated region (5' UTR) situated downstream from the PBS and upstream from the gag initiation codon. A specific stemloop structure in the 5' UTR facilitates synthetic RNA dimer formation in vitro through intermolecular `kissing' of conserved palindromic loop motifs (Clever et al., 1996
; Fossé et al., 1996
; Girard et al. , 1995
; Haddrick et al., 1996
; Laughrea & Jetté, 1994
; Paillart et al., 1996b
, 1997
). Within a 5' UTR recombination window, this kissing- loop sequence is a hotspot for nascent minus-strand DNA template switching between vector donor RNA and endogenous virus-derived acceptor RNA templates (Mikkelsen et al., 1996
, 1998b
). Although the kissing-loop interaction may constitute only one of several intermolecular interactions required for RNA dimerization in vivo (Berkhout & van Wamel, 1996
; Clever & Parslow, 1997
; Haddrick et al., 1996
; Laughrea et al., 1997
; Lear et al., 1995
; Paillart et al., 1996a
; Sakuragi & Panganiban, 1997
; Tch énio & Heidmann, 1995
), this result proposes that the kissing-loop sequence induces reverse transcriptase-mediated homologous recombination and raises the question of whether kissing-loop-defective viruses are less recombinogenic within the 5' UTR dimerization and recombination window. Recent findings suggest that the frequency of recombination elsewhere in the genome, within the coding regions of different human immunodeficiency virus type 1 (HIV-1) subtype RNAs, is not significantly affected by a lack of kissing-loop homology between interacting viral RNAs (St Louis et al., 1998
).
We have in previous work studied retrovirus recombination by a forced recombination approach based on the interaction between replication-defective MLV-based vectors and an MLV-like endogenous retrovirus (MLEV), which is encapsidated into virus particles derived from murine fibroblast-derived packaging cells (Mikkelsen et al., 1996 , 1998a
, b). This approach, based on single-cycle vector replication of PBS-defective vectors, allows for studies of template shifting events within the primary dimerization region, enabling us to study if alterations and nonhomologies within the primary packaging and dimerization region influence local recombination processes. Here, we examine putative genetic interactions between vector RNAs and copackaged ERV RNAs derived from MLEV and the VL30 retroelement family. Akv and MLEV harbour highly similar packaging and terminal repeat (R) sequences (Mikkelsen 1998a
, b
), whereas members of the VL30 family contain packaging and R sequences that are not related to the corresponding regions of Akv (Adams et al., 1988
; Hodgson et al., 1990
). We describe modes of genetic interactions that take place in spite of dissimilarities in parts of the genome involved in intermolecular RNA recognition, encapsidation and strand transferring during reverse transcription.
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Methods |
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Cells, transfections and virus infections.
-2 packaging cells (Mann et al., 1983
) and NIH 3T3 target cells were grown in Dulbecco's modified Eagle medium with Glutamax-1 supplemented with 10% newborn calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37 °C in 90% relative humidity and 5·7% CO 2. Transfections of
-2 cells and selection for stably integrated vectors have been described previously (Mikkelsen et al. , 1996
). Briefly, 10 µg vector plasmid DNA was transfected into
-2 cells seeded at 5x103 cells/cm 2 on the day before transfection. Two days after transfection, G418-containing medium was added to select for stably integrated vectors. G418-resistant colonies appearing after 12 days of selection were pooled. To measure transductional efficiencies, viruses were harvested from medium left on confluent cultures of stably integrated producer cells for 24 h. Virus-containing medium was centrifuged, filtered, serially diluted and transferred to recipient cells (NIH 3T3 cells seeded at 5x103 cells/cm2 1 day prior to infection) in the presence of Polybrene (6 µg/ml). Selective medium was added 2 days after infection and resistant colonies were counted, individually isolated and expanded after 10 days of G418-selection.
Proviral DNA sequence analysis.
Genomic DNA from G418-resistant clones was prepared as previously described (Lund et al., 1993 ). Sequence analysis of individual transduced vector sequences was performed on a PCR product encompassing part of the 5' LTR, the PBS, the 5' UTR and the upstream part of the neo gene. PCR was performed with the following oligonucleotides: ON1 (5' TTCATAAGGCTTAGCCAGCTAACTGCAG 3'), matching Akv MLV positions 78387865 (Van Beveren et al., 1985
), and ON2 (5' GGCGCCCCTGCGCTGACAGCCGGAACAC 3'), matching neo positions 16561683 (Beck et al., 1982
). The resulting PCR product was sequenced by use of an upstream primer (ON3, 5' TCCGAATCGTGGTCTCGCTGATCCTTGG 3') matching Akv MLV positions 6996 (Van Beveren et al., 1985
), and for relevant clones using a downstream primer (ON4, 5' CTTCCTTTAGCAGCCCTTGCGC 3') matching neo positions 12231244 (Auerswald et al., 1981
). The 3' LTR of transduced proviruses harbouring the originally mutated PBSUmu was PCR-amplified with a primer (ON5, 5' GGGACCTTGCACAGATAGCGTG 3') matching neo positions 30083029 and a primer (ON6, 5' AATGAAAGACCCCCGAGG 3') specifically recognizing MLEV/VL30 molecular markers within U5 (a region corresponding to positions 127144 in AkvMLV; Van Beveren et al., 1985
). Amplified fragments were sequenced with ON6.
Generation and PCR-based screening of colony pools.
In some experiments, colonies of recipient cells appearing after infection and subsequent G418-selection were pooled to allow for a PCR- based screening for recombinant proviruses among a large number of transduced proviral sequences. Genomic DNA was prepared from individually expanded colony pools each obtained by pooling of all G418- resistant colonies (on average 25 colonies for PBSUmu244 and nine colonies for PBSUmu
KL) obtained on a single plate. In PCR amplifications specific for AkvMLEV and AkvVL30 recombinants, the neo-specific ON2 was used together with ON7 (5' GTCTTTCATTTGGAGGTCCCA 3'), matching MLEV-derived PBSGln (Mikkelsen et al., 1996
), and ON8 (5' TGGTGCATTGGCCGGG 3'), matching mouse VL30-derived PBSGly (Adams et al., 1988
). ON9 (5' GCCCGGGTACCCGTATTC 3') and ON10 (5' GCCCGGGTACCCGTATTC 3'), specifically recognizing PBSUmu and an MLEV marker within R (Mikkelsen et al., 1998a
), respectively, were used to screen for AkvMLEV recombinants that harboured PBSUmu. ON11 (5' TCAGACACTCAAGTCCCGGGAC 3'), matching VL30 U5 positions 449470 (Adams et al., 1988
), was used together with ON5 to specifically amplify 3' LTRs harbouring VL30- derived sequences.
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Results |
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Recombinational rescue of PBS- and -modified vectors detected by single-colony analysis
To examine the effect of altering on forced recombination with endogenous viral RNA derived from MLEV and VL30 retroelements, the PBSPro sequences of wild-type, VL30
-harbouring and kissing- loop-deficient vectors were modified to PBSUmu, designed to be an unlikely match for any known tRNA molecule (Mikkelsen et al., 1996
). PBSUmu vectors were stably transfected into
-2 cells and viruses transferred to NIH 3T3 cells. We have previously found that the replication efficiency of PBS-modified vectors is reduced five orders of magnitude as compared to the wild- type (Mikkelsen et al., 1996
). It was therefore found less relevant to study transduction of a
-deficient vector harbouring severe modifications also within the PBS.
As expected, the transduction efficiency was strongly reduced for all PBS/-modified vectors (Fig. 2
). We previously found that the mutated and defective PBS in vectors similar to PBSUmu244
in 32 out of 60 analysed transduction events was repaired during transduction to perfectly match the 3'-end of a glutamine tRNA primer (Mikkelsen et al., 1996
). Sequence analysis of the PBSGln-harbouring proviruses demonstrated that PBS-modified vectors were transduced through initial priming of cDNA synthesis on a copackaged PBSGln-containing MLEV transcript. Following interstrand minus-strand strong-stop transfer and continued minus- strand DNA synthesis through the selective marker gene, template switching was selectively observed during minus-strand synthesis within the 5' UTR to obtain the perfect PBSGln complementarity facilitating second-strand transfer (Fig. 3a
).
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According to the generally accepted model for reverse transcription (Gilboa et al., 1979 ), R and U5 regions copied during minus-strand strong-stop DNA synthesis are transferred to the 3'-end of the genome and eventually duplicated to constitute R and U5 of both proviral LTRs. In agreement with this scheme, the composition of R and U5 in the transduced proviral 3' LTR directly demonstrates the identity of the viral RNA used as template for initiation of reverse transcription. The remaining proviruses which harboured the original mutated PBSUmu and, thus, were negative for template switching in the 5' UTR, were analysed by PCR to delineate the origin of sequences in the 3' LTR. Amplifications were performed with primers matching neo and specific MLEV/VL30 U5 genetic marker positions, and the resulting amplified 3' LTR fragments were subsequently sequenced. For PBSUmu244
, PBSUmu
KL and PBSUmu436VL30
1 of 16 (a2), 3 of 7 ( b2) and 2 of 10 (c2) analysed proviruses, respectively, were found to harbour sequences of MLEV origin in the 3' LTR (Fig. 2
). These recombinant PBSUmu- containing proviruses were generated by initiation of minus-strand DNA synthesis on MLEV, interstrand minus-strand transfer, continued minus- strand synthesis through the marker gene and read-through of the mutated PBS (Fig. 3c
, d
). We have previously shown that second-strand transfer in this scenario is mediated by complementary RU5 sequences (Mikkelsen et al., 199a
).
For each vector construct, several transduced proviruses were found not to harbour sequences of MLEV or VL30 origin (Fig. 2, a3
, b3
and c3
), indicating that these proviruses were transduced through as yet unknown pathways that may not necessarily involve specific genetic interactions with heterologous ERVs. The frequency by which recombination-dependent and -independent transduction pathways appear is strongly influenced by the expression levels of vector and ERV RNAs in the packaging cells and may therefore vary in independent experiments. In none of the cases analysed had
-modified vector constructs recombined with a copackaged
- deficient packaging construct RNA as we have previously seen for vectors harbouring the entire 476 bp Akv 5' UTR (Mikkelsen et al., 1998b
).
Rare recombination events detected by PCR screening of colony pools
Since the VL30 -based PBSUmu vector may be rescued through 5' UTR minus-strand recombination with endogenous VL30 (Fig. 3b
), we next set out to elucidate whether Akv
-based vectors (PBSUmu244
and PBSUmu
KL) in rare cases can be rescued through genetic interactions with copackaged endogenous VL30 RNA. To this end, we performed multiple transduction series of Akv-derived vectors harbouring PBSUmu and subsequently pooled all G418-resistant NIH 3T3 colonies appearing on individual plates. For PBSUmu244
, 31 colony pools were obtained, each generated from a single plate containing on average 25 G418-resistant colonies per plate. For PBSUmu
KL, on average nine G418-resistant colonies appeared on 27 plates (Table 1
). Genomic DNA was prepared from each colony pool and a PCR-based screening of G418-resistant colony pools was employed to detect recombination with endogenous viruses MLEV and VL30, respectively (Table 1
).
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To test for exchange of the impaired PBS of Akv -based vectors with PBSGly as a result of recombination with endogenous VL30 RNA, all colony pools were PCR-screened with primer sets matching (i) VL30 PBSGly and neo and (ii) neo and VL30 3' U5, respectively. In one case (one out of a total of more than 1000 transduction events, 31x25+27x9 colonies; Table 1
), we found evidence for 5' UTR minus-strand recombination between the kissing-loop-deficient vector and copackaged endogenous VL30 (Table 1
). Sequence analysis of the obtained 450 bp PCR-amplified fragment revealed that template switching in this case had occurred from a donor position within the Tn5 fragment 141 nucleotides upstream from the neo start codon to an acceptor site within VL30 153 nucleotides downstream from the glycine PBS (Fig. 4
, left panel). Transfer of the nascent minus strand coincided with a 2 nt sequence identity between donor and acceptor templates. We conclude from this observation that recombination within the 5' UTR may be detected, although a combined effect of heterologous packaging and dimerization signals, distinct Akv and VL30 R regions, and lack of profound sequence identity within the 5' UTR make this type of VL30-based recombinational repair less frequent.
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Discussion |
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In single-cycle vector replication, deletion of the MLV kissing-loop leads to a 70- and 20-fold titre reduction for PBSPro and PBSUmu vectors, respectively. Previous studies of RNA dimer formation have suggested that the kissing stemloop in MLV and HIV-1 is crucial for dimerization in vitro (Clever et al. , 1996 ; Girard et al., 1995
; Laughrea & Jetté, 1994
; Paillart et al., 1994
; Prats et al., 1990
; Skripkin et al., 1994
), but not essential for virus replication in vivo (Berkhout & van Wamel, 1996
; Clever & Parslow, 1997
; Haddrick et al. , 1996
; Paillart et al., 1996a
). It remains uncertain whether RNA dimerization is crucial for packaging and whether copackaged monomers subsequently may serve as functional substrates for reverse transcription and recombination. We show here that homologous recombination between Akv and MLEV can occur within the 5' UTR despite kissing-loop nonhomology, supporting the notion that the kissing-loop, albeit serving as a recombination hotspot sequence within the 5' UTR window (Mikkelsen et al., 1996
, 1998b
), is not an essential cis-element for retrovirus recombination. Also, we demonstrate that virus genomes harbouring distinct
regions may interact through events of RU5- mediated second-strand transfer, suggesting that a lack of identity within the putative packaging and dimerization elements of heterologous recombination partners (AkvVL30 and VL30MLEV, respectively), does not impede a recombination-based exchange of genetic information at alternative sites of the two copackaged genomes. Interestingly, St Louis et al. (1998)
recently reported that nonhomology within kissing-loop sequences of different HIV-1 subtypes does not constitute a major obstacle to recombination following postulated RNA heterodimer formation, and thus that a direct looploop interaction is not a prerequisite for template switching at alternative sites of the genome. If dimer formation is indeed required for RNA packaging, these and our findings may infer that intermolecular RNA interactions elsewhere in the genome outside the leader region can assist or partly assume a heterodimerization function, facilitating in the present report Akv vector recombination with MLEV or VL30. Previous work with HIV-1- and MLV-based systems seems to support this notion (Berkhout & van Wamel, 1996
; Tchénio & Heidmann, 1995
).
One single event of 5' UTR minus-strand recombination with endogenous VL30 was detected by PCR screening of more than 1000 transduced Akv 244 and
KL proviruses. This provirus was generated by synthesis of minus-strand strong stop on VL30, intrastrand transfer, nonhomologous switching of the nascent minus-strand to the vector, subsequent copying of neo, and nonhomologous template switching within the 5' UTR to obtain perfect PBS complementarity in second-strand transfer (Fig. 4
). The recombinant provirus was characterized by sequence analysis of PCR amplicons generated from the same colony pool in two distinct PCR reactions used in screening of multiple transduced sequences (Table 1
) and we can therefore rule out that the detected event was a result of recombination during PCR amplification. This finding thus indicates that a modification of the PBS can be overcome by a transductional repair pathway involving (i) copackaging of two heterologous RNA templates, (ii) homologous intramolecular minus-strand strong-stop transfer, and (iii) nonhomologous template switching events downstream from neo and subsequently within the 5' UTR.
Torrent et al. (1994) previously reported that a small packaging signal derived from rat VL30 facilitates efficient encapsidation of recombinant RNA into MLV particles. Insertion of non- murine heterologous packaging and dimerization sequences into retrovirus vectors may improve safety by rendering the vectors less recombinogenic. Nonhomologous minus-strand DNA transfer has previously been found to mediate recombination between distinct viruses harbouring similar packaging signals and divergent R regions (Yin et al., 1997
). In addition, vector RNAs harbouring encapsidation elements of distinct retrovirus origins have been found to interact genetically during minus-strand synthesis, suggesting that homology within the presumed primary dimerization region is dispensable for the generation of recombinant proviruses (Yin & Hu, 1997
). Expanding on these crucial observations, we show here that copackaging of
- and R-distinct retrovirus-derived sequences followed by two events of nonhomologous template switching can lead to the generation of PBS-repaired AkvVL30 chimaeric proviruses. This example of complex genetic interactions during reverse transcription illustrates how recombination between unrelated retrovirus species harbouring distinct PBS,
elements and R regions may have contributed to retrovirus evolution. In consideration of the lack of sequence similarity between Akv and VL30, any two copackaged retrovirus-derived sequences will probably be able to interact genetically and, at present, there remains an open question of whether a specific dimerization process is required for this interaction. Based on these observations, it seems unlikely that specific alterations introduced into a retrovirus vector are capable of rendering the vector nonrecombinogenic without disturbing crucial cis-acting functions required for efficient vector transduction.
The possibility exists that development and outgrowth of novel replication-competent retroviruses occur through recombination between viruses that replicate or are present endogenously in different animal species. Creation of such chimaeric viruses requires co-encapsidation of heterologous viral RNAs and subsequent recombination during reverse transcription. We believe that forced recombinational rescue of PBS- impaired retrovirus vectors constitutes a powerful tool to investigate whether retroviruses of different species interact genetically and hold the potential to form novel biologically active retroviruses. Forced recombination assays may thus be utilized to study rare genetic interactions between heterologous RNAs as well as to detect unknown ERV sequences (e.g. in human cells) that harbour functional cis- elements, some of which may constitute important sequence patches in repair of retrovirus vectors.
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Acknowledgments |
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Footnotes |
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Received 13 April 1999;
accepted 26 July 1999.