Strand transfer to the 5' part of a tRNA as a mechanism for retrovirus patch-repair recombination in vivo

María L. Carrasco1, Mogens Duch1 and Finn Skou Pedersen1,2

1 Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark
2 Department of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C, Denmark

Correspondence
Finn Skou Pedersen
fsp{at}mb.au.dk


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By screening for marker-cassette deletion mutants of a murine leukaemia virus-based replication-competent vector, two occurrences of tRNA sequence patch insertions were identified. In one of the cases, 28 nucleotides from the 5' end of tRNALys4 were inserted in the plus-strand orientation, which points to a novel strand-transfer mechanism to tRNAs during reverse transcriptase-mediated retroviral recombination.


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Recombination is a major source of genetic variability in retroviruses (Temin, 1993) due to template switching between co-packaged viral RNAs during reverse transcription (Hu & Temin, 1990). Several models have been proposed to explain genetic recombination and it has been shown that more than 98 % of retroviral recombination events occur during minus-strand DNA synthesis (Zhang et al., 2000).

Recombination events involving non-retroviral sequences were first recognized in cases where heterologous genetic information conferred a distinct phenotype on an infected cell. Thus, acutely transforming viruses harbouring sequences derived from host-cell oncogenes were isolated as a result of their ability to transform cells in culture and cause rapid tumour induction in animals (Chesters et al., 2001; Rosenberg & Jolicoeur, 1997). Such capture of non-retroviral sequences has also been modelled by studies using selectable marker genes (Stuhlmann et al., 1990). Studies of homology requirements for the generation of marker-gene-resistant proviruses during recombinogenic template switching identified proviruses containing inserted host cDNA sequence patches (Pfeiffer & Telesnitsky, 2001). Moreover, incorporation of heterologous sequences that did not confer a phenotypic trait upon the virus was also reported during non-homologous recombination analysis, where two of the recombinants harboured sequences of tRNA origin (Zhang & Temin, 1993).

Here we report on patch insertions associated with deletion of non-essential heterologous sequences during passage of a replication-competent retrovirus vector (Jespersen et al., 1999; Logg et al., 2001; Duch et al., 2004). Analysis of deletion mutants of a murine leukaemia virus (MLV) replication-competent vector revealed two cases where sequences of cellular origin had been inserted. In both cases, the sequences recovered were derived from tRNAs. In one case, sequences from the 3' end of a tRNA were inserted into viral RNA in an opposite orientation, compatible with a function as a primer for reverse transcriptase (RT), somewhat similar to previous cases (Colicelli & Goff, 1986; Sun et al., 2001). Interestingly, however, the other recombinant harboured sequences from the 5' end of a tRNA in the plus-orientation, being compatible with a novel template function of tRNAs in patch-repair mechanisms of RT-mediated recombination during minus-strand DNA synthesis.

An Akv-MLV replication-competent vector carrying an IRES–EGFP translational cassette inserted in the CelII recognition site in the U3 region of the 3' viral LTR (AkvU3–EGFP) (Bachrach et al., 2002) was digested with MluI and SseI restriction enzymes to eliminate the downstream Akv-MLV enhancer. The SL3-3Bi-neo enhancer (Jespersen et al., 1999) was amplified by PCR to produce a 594 bp fragment (5' primer, 5'-GTAAAGCGGCCGTACGCGTTTAATTAACAGCTAACTGCAGTAAC-3'; 3' primer, 5'-CTCTAGAGTCGACCTGCAGGGAGGAGACCCTCCCAAG-3') and digested with MluI and SseI. Subsequently, the 594 bp fragment was cloned into the AkvU3–EGFP-digested vector giving rise to the AkvSL33U3–EGFP vector, containing the SL3-3-MLV enhancer and the Akv-MLV backbone.

AkvSL33U3–EGFP was transiently transfected into BOSC 23 packaging cells (Pear et al., 1993). The BOSC 23 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10 % (v/v) fetal calf serum. Fresh medium was supplied to confluent virus-producing cells after 24 h. Cell-free virus supernatant was collected 48 h post-transfection and added together with 6 µg polybrene ml–1 to a fresh plate of NIH 3T3 cells, grown in DMEM containing 10 % (v/v) newborn calf serum. At 24 h post-infection, the infected culture was supplied with 2 µg polybrene ml–1 for 7 days and passaged when needed (first round). Supernatant from AkvSL33U3–EGFP-infected cells was harvested after 1 week and used to inoculate fresh NIH 3T3 cells. This cycle was repeated for six rounds as shown in Fig. 1. Fluorescent green cells were observed in each of the rounds as a result of EGFP expression.



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Fig. 1. Proviral genome structure and genomic stability analysis. The AkvSL33U3–EGFP vector containing the Akv-MLV backbone (dark grey boxes), the IRES–EGFP translational cassette (white box) and the downstream SL3-3 enhancer (light grey boxes) are shown at the top. BOSC 23 packaging cells were transiently transfected with AkvSL33U3–EGFP DNA. NIH 3T3 cells were infected 48 h post-transfection. Multiple rounds of infection were performed as described and DNA from the later transfer rounds was purified for PCR analysis. The PCR primers flanking the inserted translational cassette are shown (#A and #B). PCR products were electrophoresed on a 1 % agarose gel. The complete AkvSL33U3–EGFP genome, deletion variants and size markers (M) are shown. Bands containing deletion variants were isolated and purified for cloning and sequencing.

 
DNA isolated from infected NIH 3T3 cells from rounds 3–6 was purified (DNAzol; Molecular Research Center) and PCR-amplified to study marker-gene deletion variants (Fig. 2). PCR amplification of purified DNA was performed using a set of primers flanking the inserted sequence, as shown in Fig. 1: the upstream primer hybridized to the polypurine tract (PPT), 32 bases from the IRES–EGFP translation cassette (#A, 5'-GACCCCTTCATAAGGCTTAGCTGCAGAT-3') and the downstream primer hybridized to the 3' U3 region, 167 bases from the IRES–EGFP cassette (#B, 5'-TAGTGCTTAACCACAGATATCCTGTCGTTA-3'). PCR products were electrophoresed on a 1 % agarose gel. The 1500 bp band resulting from the intact AkvSL33U3–EGFP vector was detected and used to identify deletion variants. Diffuse bands with sizes below 1500 bp corresponded to the deletion variants and were isolated from the gel and purified (GFX Purification Protocol; Amersham Pharmacia Biotech). The resulting products were cloned into the pGEM-T Easy Vector (Promega) for subsequent sequencing. Colonies emerged after an overnight incubation period at 37 °C and were screened by PCR for positive clones. Sequencing analysis of the colonies was performed using primers hybridizing to the pGEM-T vector upstream and downstream of the inserted sequences (5' primer, 5'-GTTTTCCCAGTCACG-3'; 3' primer, 5'-CAGGAAACAGCTATG-3') together with a commercial PCR Reagent Mix (Amersham Pharmacia Biotech) and Taq polymerase. PCR was carried out for 25 cycles of denaturation at 95 °C for 20 s, annealing at 50 °C for 15 s and DNA synthesis at 60 °C for 1 min. We selected only those cases where heterologous sequences had been inserted at the junction site. Deletions without heterologous sequence insertions revealed by this analysis have been published elsewhere, where the influence of the RNA secondary structure of the IRES element on the RT template switching is addressed (Duch et al., 2004). Notably, the two junction sites of the IRES sequence flanking tRNA patches were both located at positions commonly found as borders for deletions without patch repair.



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Fig. 2. Recombinants harbouring tRNA sequences. (a) A recombinant harbouring sequences complementary to 43 nt of the 3' end of tRNAMet(i), excluding the CCA tail. The nucleotide sequence of the white box containing the GTT insertion is illustrated. (b) A recombinant harbouring 28 nt identical to the 5' end of tRNALys4. (c) Suggested three-template model for Clone 10 from Zhang & Temin (1993). The structure of the recombinant adapted from the original paper is shown above. The underlined sequence corresponds to the 14 bp insert from tRNALys as identified in the original paper. We performed a recent NCBI BLAST search of the recombinant sequence and found that it contained 24 bp derived from tRNALys3/5. Extra nucleotides are shown in white boxes. The hygromycin-resistance gene, tRNALys3/5 and the MLV LTR are depicted as the three templates involved in recombination with complementary nucleotides connected as possible sites for RT template switching.

 
Sequence analysis of AkvSL33U3–EGFP revertants identified two recombination events containing insertions of tRNA sequences. Forty-three nucleotides from the 3' end of tRNAMet(i) were found inserted in the minus orientation, together with a 3 nt insert, GTT, of unknown origin, as shown in Fig. 2(a). Cases of incorporation of the 3' end of tRNAs in the minus orientation have been reported previously (Colicelli & Goff, 1986; Sun et al., 2001). In these cases, the products may result from aberrant reverse transcription reactions using the tRNA-primed products. Our finding of the tRNAMet(i)-harbouring recombinant may also have resulted from priming of RT by this tRNA, although we cannot present a model that satisfactorily explains the final structure of the recombinant. These results, together with previous observations that 3'-end sequences derived from different tRNA species are incorporated into proviruses in the minus-strand orientation, argue that these non-viral RNAs may function as aberrant primers during reverse transcription.

The other case identified contained 28 nt from the 5' end of tRNALys4 inserted in the plus orientation (Fig. 2b). The structure of the tRNALys4 recombinant suggested a function of this tRNA as a template during minus-strand DNA synthesis in RT-mediated strand transfer. A recombination model is proposed in Fig. 3 that suggests the origin of the recombinant. Minus-strand DNA synthesis initiates after annealing of the 3' end of a partially unwound tRNAPro to the primer-binding site and proceeds to the 5' end of the genomic RNA (Fig. 3a). After strand transfer from the terminal 5' to the terminal 3' repeated R sequence, the RT may reach a stretch of 5 nt, GCAUG, in the U3 region of the genomic RNA, identical to a sequence in the dihydro-uridine loop of tRNALys4. Template switching from the genomic RNA to the tRNA would take place and the 5' end of tRNALys4 would be reverse-transcribed. The last 6 nt on this 5' end, GCCCGG, are identical to 6 nt in the IRES sequence of the vector RNA, causing a new template switch for RT (Fig. 3b). The rest of the reverse transcription process follows the normal pattern of initiation of plus-strand synthesis and completion of minus and plus strands, resulting in a provirus with a patch of tRNALys4 sequences inserted in both LTRs (Fig. 3d).



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Fig. 3. Proposed model for recombination involving tRNALys4 sequences. (a) Minus-strand strong-stop synthesis primed by tRNAPro. (b) Minus-strand synthesis continuing into U3 switches template to tRNALys4 at a position of 5 nt sequence identity (CGTAC). After reverse transcription to the 5' end of tRNALys4, the RT switches back to a position of 6 nt identity (CGGGCC) in the viral RNA. (c) Plus-strand DNA synthesis is initiated and DNA copied from tRNALys4 is included. (d) Reverse transcription is completed. Viral RNA is represented by thin lines. Thick lines represent viral DNA. Dashed lines represent reverse-transcribed tRNALys4. PBS, primer-binding site.

 
The recombination events described depend on the availability of these tRNAs in the reverse transcription complex. Together with its structural roles in the virion core, the non-specific nucleic acid-binding activity of the nucleocapsid protein is consistent with the observation that significant quantities of cellular RNAs, such as rRNAs and tRNAs, are encapsidated into virions (Berkowitz et al., 1996; Cimarelli et al., 2000). It has been shown that in retrovirus particles up to 30 % of the total RNA is tRNA (Peters & Hu, 1980), compared with approximately 15 % in the cytoplasm of growing mammalian cells (Lodish et al., 1999). Since reverse transcription takes place in the target cell within the nucleoprotein complex derived from the virus particles, it is likely that a tRNA involved in recombination is carried along from the producer cells, although we cannot exclude the possibility that it might be acquired from the target cells. We recently found that tRNAs originating from the target cells can also contribute to the initiation of reverse transcription, albeit at a very low efficiency (Schmitz et al., 2002).

Interestingly, during studies of non-homologous recombination, Zhang & Temin (1993) identified insertions of sequences derived from cellular tRNAs in two cases (Clones 10 and 44). They proposed that the inserts originated from readthrough transcription followed by deletion, rather than the presence of a third template. However, based on a recent NCBI BLAST search, we have found that the structure of one of the recombinants, harbouring tRNALys3/5 sequences (Clone 10), was compatible with the mechanism for incorporation of the inserted sequence (Fig. 2c) proposed in Fig. 3. The origin of the second recombinant (Clone 44) was more complex since it contained inserts of 19 bp of the tRNAIle and 19 bp of the tRNALys from the minus and plus strand, respectively.

The results described here have identified for the first time the incorporation of the 5' end of a tRNA inserted in a plus orientation, suggesting a novel template function for tRNAs in patch-repair mechanisms of retroviruses during strand-transfer RT-mediated recombination.


   ACKNOWLEDGEMENTS
 
This work was supported by Karen Elise Jensen's Fund and the Danish Research Agency.


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Received 17 November 2003; accepted 29 March 2004.



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