Human immunodeficiency virus type 1 transductive recombination can occur frequently and in proportion to polyadenylation signal readthrough

Abstract

One model for retroviral transduction suggests that template switching between viral RNAs and polyadenylation readthrough sequences is responsible for the generation of acute transforming retroviruses. For this study, we examined reverse transcription products of human immunodeficiency virus (HIV)-based vectors designed to mimic postulated transduction intermediates. For maximization of the discontinuous mode of DNA synthesis proposed to generate transductants, sequences located between the vectors' two long terminal repeats (vector "body" sequences) and polyadenylation readthrough "tail" sequences were made highly homologous. Ten genetic markers were introduced to indicate which products had acquired tail sequences by a process we term transductive recombination. Marker segregation patterns for over 100 individual products were determined, and they revealed that more than half of the progeny proviruses were transductive recombinants. Although most crossovers occurred in regions of homology, about 5% were nonhomologous and some included insertions. Ratios of encapsidated readthrough and polyadenylated transcripts for vectors with wild-type and inactivated polyadenylation signals were compared, and transductive recombination frequencies were found to correlate with the readthrough transcript prevalence. In assays in which either vector body or tail could serve as a recombination donor, recombination between tail and body sequences was at least as frequent as body-body exchange. We propose that transductive recombination may contribute to natural HIV variation by providing a mechanism for the acquisition of nongenomic sequences.

FIG. 1.

HIV-1 homologous transduction vector and protocol for determining template switch junctions. (A) Constitutive readthrough vector. The vector illustrated is LicPuro-äc with a downstream lacZ marked by synonymous point mutations (indicated by 10 asterisks). The 117-bp internal deletion in the body lacZ is indicated by a filled box and designated licZ. Ψ, packaging signal; MA, matrix coding sequence with frameshift mutation; RRE, Rev-responsive elements; Pcmv, cytomegalovirus immediate-early promoter; lacZ, β-galactosidase gene; Psv40, SV40 early promoter; puroR, puromycin resistance gene. (B) Transductive recombination. A predicted template switch pattern for the generation of a blue provirus is shown. PCR1 to PCR4 indicate the products used for the analyses described below. (C) Restriction pattern for a white proviral clone (left) and a blue proviral clone (right). Each of the four PCR products was digested with the indicated restriction enzymes. Undigested DNA was loaded as a size control (lane −) for each sample. Note that the sense primer for PCR1 and the antisense primer for PCR4 were located in 5′- and 3′-terminal regions of lacZ that were absent from the tail. All digestion products of each PCR product in the left panel retained the size of the undigested control, indicating that none of the tail genetic markers was present and that this was a nonrecombinant clone. In the right panel, most PCR products remained undigested except for those in the HindIII and KpnI lanes of PCR2. Digestion with these indicated the presence of tail-derived sequences, demonstrating that the provirus had the structure of the last of the two-crossover blue clones represented in Fig. 2B. M, DNA size marker pUC19/ApaLI+pUC19/HaeIII.

FIG. 2.

Schematic representation of template switch patterns from restriction and sequencing analyses. (A) Schematic drawing of one of the white clone products with four crossovers. Origins of regions are indicated by shading. Uncolored boxes represent regions from the body allele, and shaded regions were derived from the tail lacZ. Short vertical lines mark restriction sites. (B) Profiles for 32 blue clones. (C) Profiles for 71 white clones. Each horizontal bar represents the region of lacZ from an individual proviral clone that was characterized by restriction analysis, and for some, by sequencing. Deletions are shown as filled (black) regions. For the final seven white clones, the clone numbers used in Fig. 4 are indicated to the right.

FIG. 3.

Frequency of tail lacZ allele sequences in proviral clones and distribution of template switch sites. (A) Population-wide tail allele frequency at various intervals, based on restriction analysis and calculated as described in Materials and Methods. (B) Schematic representation of tail and body lacZ alleles. The deleted regions of the tail and body alleles are shown as filled (black) boxes. The 10 genetic markers are indicated with short vertical lines. The interval indicated by “2” exemplifies how intervals relative to the body and tail lacZ alleles were defined to span genetic markers. (C) Distribution of crossovers, separated into entering and leaving points and localized to intervals as defined below. The average frequencies of template switching per 100 bases are indicated. (D) Template switch intervals, categorized as either entering or leaving points in relation to which lacZ allele was used as the template switch acceptor (see text for details). The positions of the 11 intervals relative to the body and tail lacZ alleles are exemplified by interval 2.

FIG. 4.

Structures of the seven aberrant proviruses. Shaded regions represent intervals from the tail lacZ; vertical lines mark restriction sites. Deletions are represented by disruptions of horizontal lines, with their lengths approximately to scale. Sequence alignments of the donor sequence (bottom sequence), the sequence present in the proviral clone (center), and the acceptor sequence (top) for deletion junctions are shown under each proviral structure. Donor and acceptor sequences that match that of the provirus are shown in bold; any homology between the donor and the acceptor at the transfer site is boxed; 3′ R sequences are underlined. The structure of a nonrecombinant white clone is shown at the top. Clones 6 and 62 differ in lacZ region crossovers but share the features of a reverse-transcribing tail beyond the lacZ portion into the 3′ R and a rejoining body using the same seven bases of donor-acceptor identity. Clone 92 is a nonhomologous recombinant between the tail region upstream of the lacZ sequences and the body, with a single base identity at the junction. Clones 64 and 20 have deletions with insertions. For clone 64, the insertion was 20 bases from the U5-PBS junction; for clone 20, the insertion was a 34-base homopolymeric A region followed by 4 bases of unknown origin. Clones 15 and 70 share a unique deletion that lacks junctional homology but differ in that clone 70 possesses a three-LTR structure.

FIG. 5.

HIV-1 polyadenylation signal readthrough and readthrough RNA-related recombination. (A) Proviral structure, lacZ allele ratio from RNase protection assay, and blue/white ratio for vectors used to determine polyadenylation readthrough. Listed from the top downward are the wild-type lacZ vector (LacPuro), the constitutive readthrough vector (LicPuro-äc), the natural readthrough vector [LicPuro(pA)äc], and the double pA control vector [LicPuro(2pA)äc]. Arrows indicate the positions of pA signals within the vector. The RNase protection ratios of tail to body lacZ alleles and recombination rates are listed to the right. (B) RNase protection assay with viral RNAs from vectors shown in panel A. Probe, undigested probe. Signals corresponding to the protected body and tail lacZ alleles are indicated by “lic” and “-ac-,” respectively; the LacPuro body contains uninterrupted lacZ. Mock, medium from cells transfected with helper plasmid only.

FIG. 6.

Comparison of body and readthrough tail sequences as participants in patch repair. (A) Schematic representation of how template switching with readthrough RNA during reverse transcription could yield a puromycin-resistant vector with functional lacZ. Note that the donor RNA template contains two copies of the internal portion of the lacZ gene, either of which could serve as a patch repair donor. Patch repair with the tail lacZ allele is shown. The approximate locations of the first- and second-round PCRs for amplifying pooled proviral DNAs are shown beneath the acceptor template. (B) Chromatograms from sequencing of PCR products from pooled genomic DNA (pooled product), from plasmid containing the body lacZ allele only (body), from plasmid containing the tail lacZ allele only (tail), and from HIV ac-äc plasmid DNA (equimolar body + tail). Arrows indicate the nucleotide positions that differed between the body and tail lacZ alleles.