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. 2007 Nov;81(22):12156-68.
doi: 10.1128/JVI.01591-07. Epub 2007 Sep 5.

Effects of identity minimization on Moloney murine leukemia virus template recognition and frequent tertiary template-directed insertions during nonhomologous recombination

Nisha K Duggal  1 , Leslie Goo, Steven R King, Alice Telesnitsky
Affiliations

Effects of identity minimization on Moloney murine leukemia virus template recognition and frequent tertiary template-directed insertions during nonhomologous recombination

Nisha K Duggal et al. J Virol. 2007 Nov.

Abstract

Homology requirements for Moloney murine leukemia virus recombination were addressed in this study by monitoring titer defects observed when acceptor/donor template identity lengths were systematically reduced. Recombination acceptors with at least 16 contiguous bases of donor template identity were recognized as efficiently as longer acceptors. In contrast, a sharp 1-log titer drop was observed for an acceptor of only 15 bases long, with an additional 1-log titer decline for an 8-base acceptor and further decreases for shorter acceptors. Eighty-three independent nonhomologous recombination products were sequenced to examine recombination template selection in the absence of significant sequence identity. These replication products contained a total of 152 nonhomologous crossover junctions. Forced copy choice models predict that forced nonhomologous recombination should result in DNA synthesis to the donor template's 5' end, followed by microidentity-guided acceptor template selection. However, only a single product displayed this structure. The majority of examined nonhomologous recombination products contained junction-associated sequence insertions. Most insertions resulted from the use of one or more tertiary templates, recognizable as discontiguous portions of viral or host RNA or minus-strand DNA. The donor/acceptor template microidentity evident at most crossovers reconfirmed the remarkable capability of the reverse transcription machinery to recognize short regions of sequence identity. These results demonstrate that recruitment of discontiguous host or viral sequences is a common way for retroviruses to resolve nonhomologous recombination junctions and provide experimental support for the role of splinting templates in the generation of retroviral insertions.

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Figures

FIG. 1.
FIG. 1.
Forced recombination assay. (A) Two-vector recombination assay as described in reference . Newly synthesized DNA undergoes a recombinogenic strand transfer from the 5′ end of an RNA donor (D) template to the 3′ end of a distinct RNA acceptor (A) template. PRSV, Rous sarcoma virus promoter; pbs, primer binding site; ppt, polypurine track. (B) RIO template. DNA is shown as a complete circle. The RIO template contains a single, modified, central LTR (ΔU U5). An SV40 promoter-driven puromycin resistance gene containing an artificial intron is separated into 5′ (puro-, in-) and 3′ (-tron, -mycin) portions. Homologous D and A regions are designated within the intron. (C) Recombination during reverse transcription using RIO vector. Upon reaching the 5′ end of the donor template, the newly synthesizing DNA is forced to undergo a recombinogenic strand transfer to a region of sequence identity within the acceptor template. After completing plus-strand synthesis and transfer, the final provirus resembles a retroviral provirus. The puromycin resistance gene and intron are reassembled, and two LTRs flank the provirus. Ψ is the packaging signal, and PSV40 indicates the SV40 late promoter. Primers shown in the bottom row are those used to analyze products as for Fig. 2D.
FIG. 2.
FIG. 2.
One-base mismatch extension by RIO vector. (A) Experimental scheme. (B) 30TM acceptor. Newly synthesized DNA transfers from a donor template to an acceptor template with 30 bases of identity. If the DNA transfers at any position within the 30 bases of identity on the donor template, an AscI site will be generated in the provirus. (C) 1TMM acceptor. The 1TMM acceptor contains 29 bases of identity and 1 mismatched base. If DNA transfers from the 5′ end of the donor and the mismatch extends, an AscI site will be generated. If the DNA transfer occurs prior to the template end and no mismatch extension occurs, the template base will be incorporated and no AscI site will be formed. Acceptor region abbreviations are given in the legend to Fig. 1. (D) PCR amplification of puromycin resistance gene from pooled proviral DNA by use of primers indicated in Fig. 1C and analysis with AscI digestion. pRSVpuro, a vector containing an intronless puromycin resistance gene amplified with primers SF304 and MK204. RIO vectors, with their artificial introns, yield a longer amplification product. PCR products from 30TM and 1TMM, undigested and digested with AscI, are shown. Alternate targets that appear in the 1TMM product pool are indicated. puro, puromycin.
FIG. 3.
FIG. 3.
Minimal sequence identity required for efficient and accurate recombinogenic strand transfer. (A) Acceptor sequences. The series of stepwise shortened acceptors from 27 bases to 0 bases of identity at the engineered acceptor position. (B) Titer/RT for 30 to 0 bases of identity vectors, determined as described in Materials and Methods. Note that the titer in the 5TM column, indicated with an open circle and connected to dashed lines, is the titer for a 5TM vector variant in which the major alternate target sites were eliminated by mutation. This alternate 5TM vector was generated to assess titers in the absence of the major alternate targets but is not used elsewhere in this report. (C) PCR products of pooled proviral products of the limited identity vectors, with or without AscI digestion. Product mobilities are indicated at left; some alternate target products are indicated at right. puro, puromycin.
FIG. 4.
FIG. 4.
Forced nonhomologous recombination. (A) RIO template containing a human sequence nonhomologous acceptor. The acceptor region included a 590-base fragment of human chromosome 14 that contains no region of identity greater than 3 bases with the 5′ end of the donor. Note that polyadenylation signal readthrough resulted in the appendage of RNA copies of plasmid backbone sequences to a subset of the vector RNAs' 3′ ends. Acceptor region abbreviations are given in the legend to Fig. 1. (B) Vector titers determined as described in Materials and Methods. 30 represents titers for the 30TM vector; human indicates humanTM values; 0 indicates the value for 0TM vector. (C) PCR amplification of cell DNA from individual puromycin resistance cell clones. The amplification product of a correctly targeted clonal 5-base acceptor product is shown, as are PCR products from representative individual humanTM product cell clones. The leftmost two lanes contain radiolabeled size standards. puro, puromycin.
FIG. 5.
FIG. 5.
Categories of nonhomologous recombination products. (A) Forced copy choice nonhomologous recombination product. DNA synthesized to the 5′ end of the donor template transfers to an acceptor template position with primer terminal microidentity. (B) Premature jump. DNA synthesis transfers from the donor to the acceptor template before reaching the 5′ end of the donor template. (C) Incorporation of tertiary template sequences. Nascent DNA transfers from the donor template to a tertiary template before subsequent transfer to the acceptor template. Illustrated here, the tertiary template used is a segment of polyadenylation signal readthrough RNA. Acceptor region abbreviations are given in the legend to Fig. 1.
FIG. 6.
FIG. 6.
Spectra of donor departure and acceptor entry sites observed in analyzed clonal products. (A) Schematic representation of humanTM vector, including polyadenylation readthrough region. Numbers at top indicate map positions on the RNA and indicate that the schematic is not to scale. Note that polyadenylation signal readthrough resulted in the appendage of RNA copies of plasmid backbone sequences to a subset of the vector RNAs' 3′ ends. (B) Compendium of donor departure and acceptor entry sites for all 83 nonhomologous recombination products. Diagrammed as in Fig. 5C. Each open circle indicates a simple recombination donor departure or acceptor entry site; each black bar indicates a corresponding site for a complex recombinant. Acceptor sites for 20-base intervals are binned; stacked circles and/or bars indicate multiple entries into a single acceptor site within the interval. The RNA strand drawn in the center of Fig. 6B represents splinting templates. Crossover site locations for those recombinants that used sequences on the same or copackaged vector RNA are shown. Horizontal lines above the splinting template indicate the locations of splinting RNA template segments; lines below indicate splinting DNA templates. Each horizontal line represents a separate tertiary template segment. Each encompasses the template segments used, but these are not precisely to scale. polyA site, polyadenylation site. Acceptor region abbreviations are given in the legend to Fig. 1.
FIG. 7.
FIG. 7.
Retrospective alternate model for generation of single-base insertion plus flanking hypermutations. Based on nonhomologous recombination product 72.P8 in reference . (A) Model proposed in reference . A single-base insertion occurs during nonhomologous template switching, followed by junction-associated hypermutation by RT as it copies the acceptor template. (B) Splinted recombination model for single-base insertion. RT uses 2 bases of microidentity to switch to the tertiary template and then 6 bases of microidentity to switch back to the acceptor template. This model calls for one base substitution between the candidate splinting template, identified by BLASTn as GenBank accession no. V01204.1, which mapped to the U3 region of a spleen necrosis virus. Note that TK gene expression in the vector used (40) was driven by a spleen necrosis virus promoter, and thus it seems likely that the apparent 1-base insertion in a nonhomologous crossover junction here arose via splinting recombination with a distal portion of the vector RNA.
FIG. 8.
FIG. 8.
Properties of crossover junctions and splinting templates. (A) Distribution of junctional microidentity lengths. Black bars represent identities observed at simple recombinants' crossover junctions; hatched bars are complex recombinants' junctional microidentities; und represents junctions for which identity lengths were not determined because a template segment was not identified. (B) Splinting (3°) templates' lengths. Hatched bars indicate minimal template lengths for inserts with unidentified templates (e.g., the length of the insert, whereas for identified templates most included flanking microidentity residues); black bars represent assigned template segments. (Bottom) An expanded view of template length distribution for shorter insert segments shown at top.
FIG. 9.
FIG. 9.
Model for RT interactions during acceptor invasion-mediated recombination. (A) RT (represented as a peanut-shaped object) after reaching the 5′ end of the donor template. The figure represents the distance between RNase H and DNA polymerase active sites as 15 bases; donor template cleavage is represented with a dashed line; the invading acceptor is shown as a gray line. (B) RT translocated after secondary RNase H cleavages. The acceptor template is shown base pairing with nucleotides 9 to 13 from the 3′ end of the nascent pretransfer DNA. Models for acceptor invasion call for subsequent branch migration to subsequently realign the primer strand 3′ end onto the acceptor template (5).
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