Extended minus-strand DNA as template for R-U5-mediated second-strand transfer in recombinational rescue of primer binding site-modified retroviral vectors

Abstract

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.

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 tRNA_i^Met 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). Ψ-2 (30), ΩE (33), and NIH 3T3 cells were cultured, and transfections and virus infections were performed as previously described (27, 32).

FIG. 2

Origin of nucleotide sequences in 5′ and 3′ LTRs of transduced vectors harboring the modified PBS. (A) 5′ and 3′ LTR sequences of four transduced proviruses containing the original nonfunctional PBS. P3 and T1.2 contained PBS-XXX, whereas KL#19 and 33E harbored PBS-UMU and PBS-Met(i)int, respectively. Akv and MLEV sequences are shown at the top and bottom of the alignments, respectively. The U5 and PBS of the MLEV sequence have been determined by various PCR-based sequence approaches as previously described (32); the sequences of MLEV R and U3 were identified in an alternative series of experiments as part of chimeric 3′ LTRs in recombinant proviruses. Hence, the MLEV sequence listed in both alignments consists of R-U5 from the upstream LTR and U3 from the downstream LTR of the endogenous provirus. Nucleotides homologous to positions in Akv are indicated by hyphens; nucleotides different from those for Akv are indicated in the MLEV sequence. Nucleotide insertions in Akv and MLEV are indicated by the introduction of colons in MLEV and Akv sequences, respectively. Single nucleotide differences or clusters of differences between Akv and MLEV are underlined and designated I to XIV, as indicated below the MLEV sequence (molecular markers XII to XIV correspond to markers I to III in reference 32). U3-R and R-U5 borders are indicated above the Akv sequence. In the lower panel (3′ LTR), the 3′ flanking sequences are listed for clones P3, KL#19, and 33E. For comparison, the PBS-Gln2 sequence is listed below the flanking sequences. N, nonidentified nucleotide positions; ND, not determined. (B) Schematic representation of sequence data. All proviruses shown harbored R and U5 of MLEV origin in the 3′ LTR. Clones P3 and T1.2 harbored MLEV molecular markers IX to XIII in the 5′ LTR R and U5 regions; the 5′ LTR of KL#19 did not contain sequences of MLEV origin, whereas clone 33E contained all 5′ R-U5 MLEV markers (IX to XIV). The primers used in PCR amplification (primer sets ON1-ON2 and ON6-ON7 [indicated by black arrows]) and sequencing (primers ON3, ON4, and ON5 [indicated by open arrows]) are indicated below the P3 provirus.

FIG. 3

Models for recombinational rescue of PBS-modified vectors. (A) RT-mediated minus-strand recombination within the 5′ untranslated region. 1, initiation of reverse transcription on MLEV which harbors a functional glutamine PBS; 2, minus-strand transfer to the vector 3′ end and cDNA synthesis through the neo gene (template shifting within the 5′ UTR allows for correct PBS complementarity in plus-strand transfer); 3, glutamine tRNA copied in plus-strand synthesis prior to the second jump of reverse transcription; 4, the resulting Akv-MLEV chimeric provirus harboring the MLEV glutamine PBS. (B) RT-mediated recombination involving an R-U5-mediated second-strand transfer. 1, initiation of reverse transcription on MLEV; 2, minus-strand transfer to the 3′ end of vector RNA and minus-strand synthesis through the PBS to the 5′ end of the vector RNA; 3, generation of the 3′ R-U5 single-stranded DNA tail allowing for plus-strand transfer; 4, various Akv-MLEV molecular marker patterns that as a result may be observed in 5′ R and U5 (3′ R and U5 are strictly of MLEV origin). Dotted boxes, mutated PBS (PBS-Mut); hatched boxes, glutamine PBS (PBS-Gln); thin lines, RNA; thick lines, DNA; dotted lines, DNA synthesis prior to second-strand transfer.

FIG. 4

Models for R-U5-mediated second-strand transfer. Model I, plus-strand template shifting. Plus-strand synthesis is initiated from the polypurine tract (PPT) with complete minus-strand DNA as the template; a circular DNA intermediate is generated based on R-U5 complementarity subsequent to limited DNA duplex unwinding and plus-strand crossover within the R-U5 region. Model II, DNA duplex invasion by nascent minus strand; conventional tRNA primer removal during plus-strand strong-stop synthesis. Degradation of plus-strand strong-stop 3′-terminal sequences copied from the glutamine tRNA is required for continued plus-strand synthesis. Model III, minus-strand-mediated displacement of plus-strand template. DNA duplex unwound concomitantly with polymerization at the 3′ end of nascent minus-strand DNA. Thin and thick lines, RNA and DNA strands, respectively; arrows, ongoing polymerization; vertical dotted lines, recombinational crossover events; black dots (in model II), degraded plus-strand DNA; PBS-Mut, mutated vector PBS.