Correlated template-switching events during minus-strand DNA synthesis: a mechanism for high negative interference during retroviral recombination

Abstract

Two models for the mechanism of retroviral recombination have been proposed: forced copy choice (minus-strand recombination) and strand displacement-assimilation (plus-strand recombination). Each minus-strand recombination event results in one template switch, whereas each plus-strand recombination event results in two template switches. Recombinant proviruses with one and more than one template switches were previously observed. Recombinants with one template switch were generated by minus-strand recombination, while recombinants containing more than one template switch may have been generated by plus-strand recombination or by correlated minus-strand recombination. We recently observed that retroviral recombination exhibits high negative interference whereby the frequency of recombinants containing multiple template-switching events is higher than expected. To delineate the mechanism that generates recombinants with more than one template switch, we devised a system that permits only minus-strand recombination. Two highly homologous vectors, WH204 and WH221, containing eight different restriction site markers were used. The primer binding site (PBS) of WH221 was deleted; although reverse transcription cannot initiate from WH221 RNA, it can serve as a template for DNA synthesis in heterozygotic virions. After one round of retroviral replication, the structures of the recombinant proviruses were examined. Recombinants containing two, three, four, and five template switches were observed at 1.4-, 10-, 65-, and 50-fold-higher frequencies, respectively, than expected. This indicates that minus-strand recombination events are correlated and can generate proviruses with multiple template switches efficiently. The frequencies of recombinants containing multiple template switches were similar to those observed in the previous system, which allowed both minus- and plus-strand recombination. Thus, the previously reported high negative interference during retroviral recombination can be caused by correlated template switches during minus-strand DNA synthesis. In addition, all examined recombinants contained an intact PBS, indicating that most of the plus-strand DNA transfer occurs after completion of the strong-stop DNA.

FIG. 1

Models for retroviral recombination: forced copy choice (A) and strand displacement-assimilation (B). Viral RNA sequences are indicated by thin lines and lowercase letters, whereas viral DNA sequences are indicated by thick lines and uppercase letters. a and b represent two copackaged viral RNAs, while A and B represent DNA sequences generated from the two copackaged RNAs. Cloverleaf, tRNA primer; dotted line, template-switching event; ppt/PPT, polypurine tract. The direction of DNA synthesis is indicated by the arrows. (A) In step 1, RT uses RNA b as a template for DNA synthesis until a break in the RNA is encountered. In step 2, RT switches to use RNA a as a template for DNA synthesis. In step 3, the resulting DNA is a recombinant with one crossover event. (B) Two DNA copies are generated from the two copackaged RNAs. In step 1, an internally initiated plus-strand DNA fragment from DNA A is displaced by an upstream-initiated plus-strand DNA. In step 2, this displaced DNA is assimilated to the complementary region of minus-strand DNA B. In step 3, the resulting DNA has a mismatched region. In step 4, a recombinant with two crossovers is formed after mismatch repair.

FIG. 2

SNV-based retroviral vectors used to study recombination during minus-strand DNA synthesis. WH221 RNA is shown in black, and WH204 RNA is shown in white. Restriction enzyme sites are indicated above the viral RNAs. Δpbs, deletion of the PBS; neo, neomycin phosphotransferase gene; sa, splice acceptor site derived from reticuloendotheliosis virus strain A; hygro, hygromycin phosphotransferase B gene; ∗, inactivating frameshift mutation generating a unique NsiI restriction enzyme site; B, BamHI; C, ClaI; Sm, SmaI; M, MluI; Bg, BglII, Ns, NsiI; Nc, NcoI; C-R, ClaI followed by EcoRI; S, SacI; N, NotI.

FIG. 3

Experimental protocol for studying recombination during minus-strand DNA synthesis.

FIG. 4

The minimum number of template switches required to produce a recombinant provirus with a functional neo and a functional hygro is dependent on the type of minus-strand strong-stop DNA transfer. (A) Intermolecular transfer. (B) Intramolecular transfer. Symbols and abbreviations are the same as in Fig. 1 and 2.

FIG. 5

Strategy for mapping recombinant proviruses in cell clones. (A) Generic structure of a recombinant provirus. Above the recombinant, restriction enzyme markers are listed, and a BamHI site contained in both WH221 and WH204 is indicated beneath the recombinant. Primer sets for differential PCR analysis and the expected sizes of the amplification products are shown below the recombinant. Arrows indicate primer directions. (B) Representative differential PCR analysis of recombinant cell clones AQ2 and W12 with primer sets 1A and 1B. L; 1-kb ladder. (C) Restriction enzyme digestion analysis of AQ2 DNA amplified with primer set 1A. (D) Restriction enzyme digestion analysis of W12 DNA amplified with primer set 1B. U, undigested DNA. The 5′ structure of each recombinant proviral DNA derived from the analysis is illustrated below the restriction enzyme mapping gel in panels C and D. Symbols and abbreviations are the same as in Fig. 1 and 2.

FIG. 6

Restriction enzyme maps of recombinant proviruses. Maps are shown with an intermolecular minus-strand DNA transfer (A) and an intramolecular minus-strand DNA transfer (B). WH221-derived sequences are shown in black, whereas WH204-derived sequences are shown in white. Restriction enzyme sites are listed above each genotype, and arrows indicate the selectable markers in neo and hygro. The number to the left of each genotype represents the number of crossovers, and the number to the right indicates the number of recombinants of the same genotype observed among the 47 recombinant proviruses analyzed. Restriction enzyme abbreviations are as given in the legend to Fig. 2.