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Previous Article | Next Article 
J Virol, March 1998, p. 2519-2525, Vol. 72, No. 3
Department of Molecular and Structural
Biology1 and
Department of Medical
Microbiology and Immunology,2 University of
Aarhus, DK-8000 Aarhus, Denmark
Received 14 August 1997/Accepted 13 November 1997
We have previously demonstrated recombinational rescue of primer
binding site (PBS)-impaired Akv murine leukemia virus-based vectors
involving initial priming on endogenous viral sequences and template
switching during cDNA synthesis to obtain PBS complementarity in
second-strand transfer of reverse transcription (Mikkelsen et al.,
J. Virol. 70:1439-1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of
different structures, suggesting that PBS knockout vectors may be
rescued through initial priming on endogenous virus RNA, read-through
of the mutated PBS during minus-strand synthesis, and subsequent
second-strand transfer mediated by the R-U5 complementarity of the plus
strand and the extended minus-strand DNA acceptor template. Mechanisms
for R-U5-mediated second-strand transfer and its possible role in
retrovirus replication and evolution are discussed.
Retroviruses harbor a diploid
single-stranded RNA genome which constitutes the source for generation
of double-stranded DNA by reverse transcription. DNA synthesis is
initiated from the 3' end of a host-derived tRNA matching the
18-nucleotide primer binding site (PBS) located downstream from the U5
region. The resulting minus-strand strong-stop DNA is in turn
transferred to the 3' end of either one of the copackaged
RNAs. This first-strand transfer (or jump) is facilitated by the
complementarity of the terminal R regions and furthermore by the
reverse transcriptase RNase H-mediated degradation of 5' R and U5 RNA
in the RNA-DNA hybrid generated (3, 11, 29, 40, 57).
Minus-strand DNA molecules shorter than strong-stop DNA (designated
weak-stop DNA) are occasionally generated by premature termination and
strand transfer (2, 20, 21, 26, 44, 58, 67), indicating that
R-region homologies shorter than the entire length of R are sufficient
for transfer to occur. After transfer of weak- or strong-stop DNA,
minus-strand DNA synthesis advances toward the 5' end of the RNA
template. Plus-strand synthesis, which is primed from a purine-rich RNA
fragment upstream from the U3 region (18, 28, 45, 46), is
believed to proceed until the first modified tRNA nucleotide is
reached, leading to regeneration of the PBS matching the primer tRNA
(48, 56). The tRNA primer is subsequently removed by RNase
H-mediated degradation (5, 37, 50, 52). The complementarity
of the plus-strand 3' PBS obtained by copying the tRNA primer and the
minus-strand 3' PBS mediates the second-strand transfer (1,
10), and, finally, plus- and minus-strand syntheses are
completed.
Except for the apparent requirement for PBS complementarity in the
second jump of reverse transcription (10, 56, 58), little is
known about the transfer reaction and the acceptor template involved.
Clearly, correct strand transfer cannot occur before complementary
sequences have been copied during plus- and minus-strand synthesis
(58). In theory, such sequences may include not only the PBS
but also non-PBS sequences (22, 23, 36, 38, 41, 42) and, in
particular, the R-U5 region upstream from the PBS. Copying of the R-U5
region during minus-strand synthesis may depend on whether minus-strand
synthesis has been initiated from both PBSs in the genomic RNA dimer
(leading to degradation of R-U5 by RNase H) and whether read-through of
the PBS is influenced by potential tRNA occupancy of the PBS.
Copackaging of heterologous viral RNAs and a subsequent high rate of
recombination during reverse transcription have been demonstrated in
numerous studies (14-17, 39, 54, 55, 59, 66, 68). Reverse
transcription-mediated recombination may involve endogenous virus-like
elements, as seen in studies of various replication-defective
retroviral mutants (6, 7, 9, 31, 32, 34, 51). Endogenous
viral RNAs found to be encapsidated in virus particles (13, 32,
43, 49) may thus serve to provide the functional sequences
required for repair of deleterious viral mutations. Such
recombinational rescue mechanisms may include template shifting during
minus- or plus-strand DNA synthesis and may be influenced by the
character of the two strand transfers of reverse transcription
(39, 59, 66).
Forced recombination of PBS-modified vectors.
In agreement
with the essential role of the PBS in initiation and completion of
reverse transcription (35, 47, 62), we previously observed a
strong restriction in transduction of Akv murine leukemia virus (MLV)
vectors with PBSs having only partial (PBS-XXX) or no [PBS-UMU and
PBS-Met(i)int] homology with the 3' end of any known murine tRNA
molecule (32). In experiments based on virus production in
NIH 3T3 cell-derived packaging cell lines (
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Extended Minus-Strand DNA as Template for R-U5-Mediated
Second-Strand Transfer in Recombinational Rescue of Primer Binding
Site-Modified Retroviral Vectors
ABSTRACT
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E) some of the
transduced proviruses were found to harbor sequences originating from
both vector and endogenous virus, suggesting that the impairment of the
PBS was circumvented in some cases by reverse transcription-mediated
minus-strand recombination with an endogenous virus containing a
functional PBS (32). Repair of PBS mutants involved
initiation of cDNA synthesis from the functional glutamine PBS of
copackaged MLV-like endogenous virus (MLEV) RNA, an interstrand
first-strand transfer followed by minus-strand synthesis through the
neo gene, and template shifting within the 5' untranslated
region (5' UTR) to allow for the interaction of complementary glutamine
PBS sequences during second-strand transfer. Hence, transduction of PBS
knockout vectors in a single-cycle transfer protocol, as delineated in
Fig. 1B, is performed under triple
selection for (i) reverse transcription initiation, (ii) second-strand
transfer, and (iii) expression of the neo gene (Fig. 1A). In
the present report, we focus on recombination-mediated transduction of
proviral sequences which have retained the original, mutationally
impaired PBS (referred to as type 2 proviruses in reference
32).
View larger version (21K):
[in a new window]
FIG. 1.
Principles of forced recombination. (A) Nonfunctional
PBS sequences introduced into Akv MLV-based vectors harboring the
neomycin resistance gene (neo). Selection for (i) initiation
of minus-strand synthesis, (ii) successful second-strand transfer, and
(iii) expression of the marker gene represents an effective selection
pressure that allows for detection of recombinational vector rescue.
(B) Experimental approach. PBS-modified vectors in a single-cycle
vector replication protocol were investigated utilizing NIH 3T3-derived
virus producer cells and NIH 3T3 target cells. Three different
PBS-modified constructs were utilized, harboring PBS sequences that
were designed to unlikely match the 3' end of any known murine tRNA
molecule (32). The PBS sequences introduced included PBS-XXX
(retaining the nucleotides complementing the tRNA CCA tail), PBS-UMU
(in which all of the wild-type PBS positions were altered), and
PBS-Met(i)int (matching an internal fragment of
tRNAiMet suggested to serve as a primer in
Drosophila copia retrotransposon replication
[19]). G418-resistant colonies were cloned and
subjected to sequence analysis in order to elucidate individual
transduction pathways. Genomic DNAs from G418-resistant clones were
prepared as previously described (27). -2
(30), E (33), and NIH 3T3 cells were cultured,
and transfections and virus infections were performed as previously
described (27, 32).
Sequence analysis of transduced PBS and LTR sequences. Transduced G418-resistant NIH 3T3 target cells from five different series of virus transfer experiments (more than 80 clones altogether) were individually screened by PCR to detect proviruses resulting from recombination of the vector with MLEV. Initially, the origin of the PBS was determined by sequence analysis of a 1.37-kb PCR fragment spanning U3, R, U5, PBS, the 5' UTR, and part of the neo gene; the proviruses harboring the original, mutated PBS were subjected to further analysis. According to the model for reverse transcription (10), the minus-strand strong-stop DNA containing the R and U5 regions copied during minus-strand strong-stop synthesis is transferred to the 3' end of the genome in the first jump of reverse transcription. Therefore, we then tested by PCR amplification and subsequent sequence analysis whether the 3' long terminal repeat (LTR) of mutant PBS-harboring subclones contained non-Akv sequences. The PCR was performed with a neo primer and a primer specifically recognizing the MLEV molecular marker XIV (Fig. 2A). Indeed, in four cases (clones P3, T1.2, KL#19, and 33E) we found the specific MLEV pattern of scattered molecular differences from Akv (Fig. 2A, lower panel). In contrast to the R and U5 regions, the U3 region was found to be identical to Akv U3. These findings indicated that transduction of at least some of the type 2 proviruses involved initiation of reverse transcription on the functional glutamine PBS of MLEV.
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Model for alternative recombinational vector rescue. In our previous work, we observed that type 1 PBS-Gln-containing proviruses are generated through 5' UTR minus-strand recombination-based patch repair of PBS-impaired vectors (Fig. 3A) (32). Based on the observations presented here, we propose that PBS-modified vectors are alternatively rescued through an initial priming on the copackaged MLEV followed by interstrand minus-strand transfer and minus-strand synthesis through the neo gene and the impaired PBS.
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R-U5-mediated second-strand transfer. According to the currently recognized model for reverse transcription of retroviral RNA (10), the PBS copied during generation of full-length plus-strand strong-stop DNA is unmasked by degradation of the tRNA template by reverse transcriptase RNase H activity (5, 10, 37, 52), thereby allowing for subsequent PBS-mediated second-strand transfer. In R-U5-mediated second-strand transfer transduction, in contrast, strand transfer does not involve complementary PBS sequences. Therefore, we were faced with the challenge of modeling unconventional second-strand transfer of a nascent or partly degraded plus strand being part of a DNA-DNA duplex to a nascent or possibly complete R-U5-extended minus-strand DNA.
Considering the structural features of the circular RNA-DNA intermediate in reverse transcription and the time course of minus- and plus-strand synthesis, we propose three distinct models for non-PBS-mediated plus-strand transfer (Fig. 4). The suggested models are based on the assumption that minus-strand synthesis is completed before plus-strand synthesis (model I), that full-length plus-strand strong-stop DNA is generated before completion of minus-strand synthesis (model II), or that strand transfer is mediated by the interaction of nascent minus and plus strands (model III). In model I, transfer of an incomplete plus-strand DNA may be the result of a reverse transcriptase-mediated template switch in which the nascent plus strand is transferred to the completed minus strand, the template for continued plus-strand synthesis. This mechanism would require exposure of R-U5 sequences by limited unwinding of the DNA-DNA duplex mediated potentially by the reverse transcriptase (8) or by the nucleocapsid protein which recently has been shown to facilitate DNA duplex melting (60). Model II, in contrast, implies that the 3' end of the nascent minus strand invades the DNA duplex containing complete plus-strand strong-stop DNA. Hence, this transfer mechanism involves partial degradation of the plus strand, allowing for continued plus-strand synthesis through the modified PBS sequence. Alternatively, the template for the growing plus strand may be displaced by the nascent minus strand, thereby mediating premature plus-strand transfer (model III). Although minus-strand-mediated displacement may also require limited unwinding of the DNA duplex, models II and III are in accordance with the fact that the invading minus strand is unmasked by RNase H degradation of the RNA template and are moreover in accordance with in vitro studies demonstrating an extensive DNA duplex displacement capacity of the Moloney MLV reverse transcriptase apparently coupled to minus-strand DNA synthesis (64). Furthermore, in agreement with models II and III, it appears from previous studies that plus-strand synthesis is initiated prior to completion of the minus strand (4, 24, 25). However, in the present study based on molecular markers within relevant regions, we cannot distinguish among the models discussed.
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tRNA removal in recombinational transduction mediated by unconventional second-strand transfer. Models I and III (Fig. 4) predict that if the primer tRNA is removed (see below), it is removed late in reverse transcription after synthesis of the complete plus strand, since RNase H specifically degrades only RNA in DNA-RNA hybrids (5) and, therefore, will not remove a tRNA that has not been copied during plus-strand synthesis.
To test whether tRNA sequences could be found as part of the integrated recombinant proviruses downstream from the 3' LTR, we performed sequence analysis of the unknown DNA flanking the downstream proviral LTR. Flanking DNA was amplified by a two-step semirandomly primed PCR approach (53). Briefly, in the initial PCR step, a specific biotinylated primer matching Akv U3 and a panel of degenerate primers were utilized in a series of PCRs performed with genomic DNA which had previously been digested with PvuI (at a unique site downstream from the Akv 5' LTR) to avoid amplification of internal proviral sequences. The PCR products were purified and utilized as templates in a second PCR in which products of the initial PCR were reamplified by using a nested U3-specific primer together with a linker-specific primer. The resulting PCR products were purified and sequenced. As shown in Fig. 2A (lower panel), we were not able to detect sequences originating from the primer tRNA in the flanking regions in any of the clones analyzed, suggesting that the tRNA had been removed correctly before viral integration. Therefore, we propose, in cases of premature plus-strand transfer (models I and III), that the tRNA primer is removed subsequent to second-strand transfer after completion of viral DNA synthesis. However, it is noteworthy that such late tRNA removal would result in the generation of a single-stranded 18-nucleotide 3' extension that may be degraded by cellular nucleases prior to integration or by the integrase during the process of integration. Support for the latter explanation comes from in vitro studies by Vink et al. (61), who found that human immunodeficiency virus type 1 substrates with single-stranded 6-deoxyribonucleotide extensions 3' of the CA sequence could be cleaved and integrated by human immunodeficiency virus type 1 integrase. It should also be noted that if our data reflect strand invasion by the growing minus strand (model II [Fig. 4]), the tRNA would be removed conventionally after completion of plus-strand strong-stop DNA.R-U5 second-strand transfer in MLV replication. The question of whether transfer of an incomplete or degraded plus strand is a frequent event in MLV reverse transcription or is seen here only as a result of a marked selection pressure remains. Since specific PBS-tRNA interactions are of major importance in MLV primer selection (27), we do not expect any tRNA to bind the modified PBS in our recombination system based on PBS nonfunctionality. Consequently, cDNA synthesis is not initiated on the vector RNA and, moreover, potential tRNA binding will not interfere with PBS read-through during cDNA synthesis. The result is a minus-strand 3' R-U5 extension generated due to the lack of RNase H-mediated RNA degradation subsequent to minus-strand strong-stop synthesis. Evidence has not been provided that both PBS sequences in a wild-type virus are bound by their matching tRNAs. Indeed, studies by Whitcomb et al. (63) have demonstrated that approximately 70% of the avian leukosis virus PBS sequences are occupied by matching tRNA primers, thus suggesting that minus-strand DNA synthesis is initiated from only part of the PBS sequences in a virus population. Therefore, we cannot exclude the possibility that interstrand minus-strand transfer in reverse transcription sometimes is followed by an intrastrand non-PBS-mediated plus-strand transfer. It was recently demonstrated that genetically distinct retroviruses having similar PBS sequences may recombine in vivo (66). Interestingly, R-U5-mediated second-strand transfer, as described in the present report, may allow for recombination of distinct retroviral species that differ within the PBS region but that have some homology within the R-U5 region. Hence, generation and transfer of incomplete plus-strand DNA, which were here selectively seen in reverse transcriptase-mediated recombinational rescue of PBS-impaired retroviruses, may play a role in retrovirus replication and evolution.
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ACKNOWLEDGMENTS |
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This work was supported by the Danish Biotechnology Programme, the Danish Cancer Society, the Novo Foundation, the Danish Natural Sciences Research Council, the Karen Elise Jensen Foundation, and contracts Biotech CT95-0100 and Biomed2 CT95-0675 of the European Commission.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular and Structural Biology, University of Aarhus, C. F. Moellers Allé, Bldg. 130, DK-8000 Aarhus, Denmark. Phone: 45 89423188. Fax: 45 86196500. E-mail: fsp{at}mbio.aau.dk.
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