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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ABSTRACT |
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MAIN TEXT |
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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 IRESEGFP translational cassette inserted in the CelII recognition site in the U3 region of the 3' viral LTR (AkvU3EGFP) (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 AkvU3EGFP-digested vector giving rise to the AkvSL33U3EGFP vector, containing the SL3-3-MLV enhancer and the Akv-MLV backbone.
AkvSL33U3EGFP 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 ml1 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 ml1 for 7 days and passaged when needed (first round). Supernatant from AkvSL33U3EGFP-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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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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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.
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ACKNOWLEDGEMENTS |
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Received 17 November 2003;
accepted 29 March 2004.
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