Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011-1250, USA1
Author for correspondence: Susan Carpenter. Fax +1 515 294 8500. e-mail scarp{at}iastate.edu
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Abstract |
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Introduction |
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To explore the mechanism of size variation in the BIV and EIAV SU proteins, we analysed the size variable regions for elements known to contribute to recombination in other viruses. The SU protein-coding region of BIV and EIAV contained two elements that may promote strand transfer and duplication/insertion events consistent with current models of RNA recombination: an AU-rich region and nucleotide sequence identity between the switching site on the donor template and the landing site on the acceptor template. Mutation of either, or both, of these elements decreased the efficiency of imprecise strand transfer in the BIV SU protein. These findings support a model of size variation in the BIV SU protein resulting from duplication/insertion events that occur during minus-strand DNA synthesis and suggest that intrinsic genetic elements may modulate the frequency of recombination in vivo.
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Methods |
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The donor template plasmid pBD6, containing the full-length variable region, was made using a forward primer containing an EcoRI site and T7 promoter sequences in addition to the BIV SU protein sequences. The reverse primer comprised nt 432450 of the BIV SU protein and a HindIII site, which allowed cloning of the amplicon into pUC19 restricted with EcoRI/HindIII. A second donor template lacking the proposed landing site, designated pBD3b, was constructed to avoid possible intramolecular strand transfer between the switching site and landing site within one donor molecule. The pBD3b donor plasmid, containing nt 260370, was amplified by PCR using primers containing 3'-terminal restriction sites, which allowed insertion into the KpnI and BamHI sites of pBluescript SK(-) (Stratagene) under the control of the T7 promoter. To introduce mutations in the AU-rich region and/or the sequence identity region, a NcoI site was introduced at nt 350 of pFSU11. A double-stranded oligonucleotide cassette, which contained the AU-region mutation (AAUAUUACCGCGGC) and/or the sequence identity mutation (AACCC
AGTCG), was synthesized to replace the wild-type XbaINcoI fragment in pFSU11. Mutated fragments were amplified and cloned into the KpnI and BamHI sites of pBluescript SK(-) using the primer pairs described above. The wild-type acceptor plasmid, pFA6, was constructed by PCR amplification and cloning of the BIV SU protein nt 220430 into pUC19 using primers containing T7 promoter sequences and either EcoRI or HindIII sites. Mutations in the acceptor templates were constructed in pUC19 using the same strategy as the wild-type acceptor except that one of the PCR primers contained the desired mutation (AACCC
AGTCG). All constructs were confirmed by DNA sequencing.
Synthesis and purification of donor and acceptor templates.
The donor and acceptor template plasmids were linearized by restriction enzymes and template RNA was transcribed with T7 RNA polymerase, according to the protocol provided with the RibMAX kit (Promega). The RNA transcripts were electrophoresed on a 6% denaturing polyacrylamide gel, excised as a single full-length transcript band, purified using the RNaid kit (BIO101) and quantified by measuring the A260 with a UV spectrophotometer.
In vitro strand transfer assay.
An in vitro strand transfer reaction was carried out at 37 °C according to the method described by Guo et al. (1997) . DNA primers BF450BC' (5' ATCCGTGTCCTCCGAGCCC 3') or SK1 (5' GCTCTAGAACTAGTGGATC 3'), complementary to either nt 430450 of the BIV SU protein or pBluescript SK(-), respectively, were 5' end-labelled with 32P, as described previously (Guo et al., 1997
). The primer was incubated with 0·2 pmol of donor template at a template:primer ratio of 1:2 at 65 °C for 5 min and gradually cooled to room temperature within 40 min (
1 °C/min). 0·2 pmol of acceptor RNA was added and incubated in a 20 µl reaction mixture containing 0·1U/µl HIV-RT (Worthington), 100 µM dNTPs and 7 mM Mg2+. Reverse transcription was initiated by the addition of dNTPs and Mg2+. The RT enzyme was the heterodimeric form containing RNase H activity. The reactions were incubated at 37 °C for 35 min, or as specified, and stopped by adding EDTA to a final concentration of 50 mM, followed by proteinase K treatment and phenolchloroform extraction. The sample was mixed at a ratio of 1:1 with denaturing loading buffer (80% formamide, 2 mM EDTA, 0·01% xylene cyanol and 0·01% bromophenol blue) and heated at 90 °C for 5 min. The sample was analysed by PAGE in a 6% gel containing 7 M urea. Following electrophoresis, the gel was dried, exposed to a phosphorimager screen and the reaction products were quantified using the PhosphorImager and ImageQuant (Molecular Dynamics) software. Strand transfer efficiency was calculated as the percentage of the sum of signal intensity of strand transfer bands versus the sum of the signal intensity for all the bands with molecular mass equal to or larger than that of the full-length primer extension product (Palaniappan et al., 1996
). All assays were done in duplicate and repeated at least three times.
Sequence analysis of strand transfer products.
Bands representing strand transfer products were excised from the polyacrylamide gel and the single-stranded DNA within the band was purified as described previously (Maniatis et al., 1982 ). The DNA was amplified either by PCR using the template-specific primers BF376 (5' TCCAGTCGTGGTGTTGCAC 3') and SK1 or by 5' RACE (Gibco-BRL). For reactions using template-specific primers, PCR amplification started with 1 cycle of denaturation at 95 °C for 1 min, annealing at 57 °C for 1 min and extension at 72 °C for 1 min, followed by 9 cycles with the same denaturing and extension conditions but with the annealing temperatures decreasing from 57 to 52 °C at 0·5 °C change per cycle. This was followed by 30 cycles of 95 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min and a final extension for 7 min at 72 °C. In some cases, 5' RACE was used to ensure amplification of products with altered or missing 3' ends. A homopolymeric tail containing dC was added to the purified minus-strand DNA product by terminal deoxynucleotidyl transferase. This modified minus-stand DNA was used as the template in a PCR using a primer containing homopolymer dG and either BF450bC' (for pBD6 product) or SK1 (for pBD3b). In all cases, the PCR products were ligated to the pGEM-T easy vector (Promega) and transformed into Escherichia coli SureCell (recB recJ) (Stratagene). Individual clones were picked and sequenced.
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Results |
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Strand transfer occurs in the hypervariable region of the BIV SU protein
To examine the role of strand transfer in BIV size variation, we used an in vitro strand transfer assay in which donor and acceptor templates were of varying length and a unique primer-binding site was present on the donor template (Fig. 2). A 32P end-labelled primer was used to initiate reverse transcription along the donor RNA template and subsequent strand transfer events resulted in a final minus-strand DNA product which was increased in length due to the additional sequences at the 3' end of the acceptor template. Both precise and imprecise strand transfer products were identifiable by their size following electrophoresis in denaturing polyacrylamide gels.
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Identification of the switching and landing sites in the imprecise strand transfer products
To confirm the identity of the higher molecular mass products observed in Fig. 4(B), the IT products from reactions containing pBD3b donor RNA were excised from the gel and amplified by PCR using primers specific for strand transfer products. Amplicons were cloned and 16 individual clones were sequenced to identify crossover sites. Three different in vitro crossover sites, designated A, B and C, were found among the 16 clones sequenced (Fig. 5A
). The switching sites were distributed along a 53 nt region in the donor template, whereas the landing sites were clustered in a specific 20 nt region. In each pair of switching and landing sites, there was 35 nt sequence identity that appeared to facilitate re-association between the nascent minus-strand, single-stranded DNA and the acceptor template. Although we cannot rule out the possibility that these recombination events could also occur during the synthesis of plus-strand proviral DNA, these findings are consistent with a model of size variation in the BIV SU protein that results from imprecise strand transfer during minus-strand DNA synthesis.
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Cis-acting elements can increase the frequency of imprecise recombination
The size variable region of BIV contains cis-acting elements that contribute to recombination in other RNA viruses. Therefore, it was of interest to consider whether these elements contribute to imprecise strand transfer in our in vitro assay. A series of mutations designed to reduce AU content and/or switching/landing site sequence identity were introduced into the donor and acceptor RNAs (Fig. 3B, C
) and tested in our strand transfer assay (Fig. 6
). Mutation of the AU-rich region on the donor template (
AU) resulted in a decrease in the efficiency of imprecise strand transfer of more than eightfold as compared to the wild-type donor RNA. The efficiency of imprecise strand transfer was also reduced by disruption of sequence identity between the switching and landing sites, as indicated by mutation of either the switching on the donor RNA (
SS) or the landing site on the acceptor RNA (
LS). A similar reduction in imprecise strand transfer was observed when the donor RNA contained both
SS and
AU mutations. These data indicated that both the AU content and the sequence identity between the switching and landing sites can contribute to the rate of imprecise strand transfer.
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Discussion |
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An in vitro strand transfer assay was used to examine cis-acting elements that promote template switching/strand transfer in the size variable region of the BIV SU protein. We observed both precise strand transfer, which does not produce size variation, and imprecise strand transfer, which leads to duplication/insertion. In the model proposed for size variation in the BIV SU protein (Fig. 1C), the nucleotide sequence identity between the switching site on the donor and the landing site on the acceptor facilitates incorrect alignment of the nascent minus-strand DNA with acceptor template. Indeed, disruption of sequence identity by mutating the donor switching site motif from AACCC to AGTCG drastically reduced the efficiency of imprecise strand transfer. However, sequence identity alone appears to be insufficient for determining the site of re-association, as demonstrated by the fact that the use of an acceptor template containing the compensatory mutation did not restore levels of imprecise strand transfer to that observed with wild-type templates. Therefore, factors in addition to base pairing are required for association of the nascent minus-strand DNA with the acceptor template. The likelihood that a stretch of nucleotides can act as the landing site for non-homologous recombination may depend on its accessibility (e.g. single-strandedness or the spatial relationship with the switching site or nascent strand). It has been reported that the landing site on the acceptor template is preferentially located in single-stranded regions, ready to base pair with the detached nascent strand during template switching (White & Morris, 1995
). The most straightforward explanation for the failure of the compensatory acceptor to restore imprecise strand transfer is that the mutation changed the folding of the acceptor template in such a way that the landing site was no longer an ideal landing site. Also, the landing site may require a spatial configuration that brings appropriate switching and landing sites into close proximity with one another. For example, a kissing-loop dimerization region within the MLV 5' leader sequence is thought to be a hot spot for recombination because of the formation of an RNA heteroduplex within this region (Mikkelsen et al., 1998
). Detailed RNA secondary structure analysis of the BIV SU protein donor and acceptor templates will help to delineate the mechanism of how the switching site is guided to the landing site.
Genetic variation, including point mutations and recombination, plays an important role in enabling lentiviruses to adapt to the changing host environment. During BIV replication in vivo, SU protein variants containing duplications/insertions appeared at 28 days post-infection, a few days after the onset of BIV-specific antibodies, and became dominant 1 year after infection (Suarez & Whetstone, 1995 ). Thus, the larger SU protein variants had a selective advantage in vivo. Approximately one-half of the recombinants generated in vitro contained in-frame insertions and each insertion duplicated a putative N-linked glycosylation site. The complex factors important in variant selection in vivo may explain why we did not get the exact duplication/insertion sequence as was observed in vivo. However, the similarity between the in vitro and in vivo recombinants with respect to the crossover site and nature of insertion indicates that all variants probably arose through a similar mechanism of recombination. Thus, both virus and host factors may contribute to the generation and selection of recombinant viruses in vivo.
The in vitro studies reported here are the first to explore the mechanism of lentivirus recombination using virus sequences that are known to undergo size variation in vivo. The finding that cis-acting elements can influence strand transfer efficiency during reverse transcription supports other studies demonstrating hot spots of retrovirus recombination (DeStefano et al., 1992a ; Klarmann et al., 1993
; Wooley et al., 1998
; Mikkelsen et al., 1998
). However, our in vitro assay system cannot fully duplicate the events of strand transfer/recombination in vivo and additional studies are needed to elucidate fully the mechanisms of genetic recombination during lentivirus infections. Notably, the only protein in our in vitro system was the heterodimeric RT. Numerous studies have demonstrated that the NC protein plays a critical role in the initiation of reverse transcription (Prats et al., 1988
; Barat et al., 1989
; Li et al., 1996
; Lapadat-Tapolsky et al., 1993
, 1997
) and can stimulate strand transfer by acting as an RNA chaperon (Allain et al., 1998
; Rodriguez-Rodriguez et al., 1995
; Guo et al., 1997
; Kim et al., 1997
; Wu et al., 1996
). Also, we used heterologous HIV-1 RT in the assay system. Although lentivirus RT proteins share some degree of identity, the inclusion of the NC protein in a homologous system will mimic virus replication in vivo more precisely. Further elucidation of genetic mechanisms of lentivirus recombination and their role in persistence and pathogenesis will aid in the understanding of virus evolution and the design of effective vaccines and antiviral therapies.
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Acknowledgments |
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Received 11 April 2001;
accepted 28 August 2001.
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