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.Retroviral transduction of cellular genes is well documented (5, 58) and was best characterized by the discovery of the cellular counterparts of the oncogenes in acute transforming retroviruses (49). These oncogenes were found embedded in viral genomes, replacing part of the viral coding sequences and rendering most acute transforming retroviruses replication defective. Scattered evidence, including a drug-resistant patient isolate and vectors studied in cultured cells, suggests the possibility of transduction by human immunodeficiency virus type 1 (HIV-1) (43, 53), but the ability of HIV to perform the specific steps required for transduction has not been addressed experimentally.Several models have been proposed for oncogene transduction (4,15,16,19,29,38,41,44,50,54,56,61). Most suggest that the fusing of host and viral sequences resulted from a series of rare events that are intrinsic to the viral replication strategy (52). One model for transduction is as follows. First, a retrovirus integrates just upstream of the sequence that will be transduced. Retroviral polyadenylation signals are leaky (18), and readthrough of such signals fuses sequences adjacent to the proviral insertion to the viral genomic RNA's 3Ј end. Such readthrough transcripts can be encapsidated and serve as templates for normal reverse transcription products whose synthesis bypasses the RNA readthrough 3Ј ends (19,55)...

The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

“…Other evidence comes from observations that recombination is fully as frequent when only minus-strand recombination is possible as when both minus-and plus-strand recombination could contribute (12,148,355,359). Another argument against significant plus-strand recombination lies in the very high frequency of minus-strand crossovers, because the template degradation which that would require precludes extensive plus-strand diploidy (221).…”

“…Additional matches so identified may reflect the inadvertent introduction of host sequences into databases by investigator error. However, one prominent class of insertions that often scores as significantly similar to host sequences is env variable region inserts (12). These N-linked glycosylation siteencoding regions in HIV-1 isolates have a G-C content very different from those of other HIV-1 genome regions and recede and reappear episodically rather than by gradual point mutation (35, 170, 363) (Fig.…”

“…Endogenous retroviruses contribute less to HIV-1 genetics because no human retroelements resemble HIV-1, and gRNAs of human endogenous viruses are not readily packaged by HIV-1 (357). However, rarely encapsidated RNAs can contribute to recombination in proportion to their prevalence (12). In addition to the "random" low-level mRNA packaging, many RNA sequences that presumably cannot heterodimerize with gRNAs are capable of directing RNA packaging in a virus species-specific manner (62,84).…”

Pseudodiploid Genome Organization Aids Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis

“…Twelve days later, puromycin-resistant colonies were fixed and stained with X-Gal (5-bromo-4-chloro-3-indolyl-␤-Dgalactopyranoside) for ␤-galactosidase activity as described previously (3). Viral vectors were produced for serial passage by transfecting pools of puromycinresistant clones with helper plasmids (pHEF-VSVG and pCMV⌬R8.2), and the resulting virions were harvested for further infections at 2 days posttransfection.…”

Effects of Identity Minimization on Moloney Murine Leukemia Virus Template Recognition and Frequent Tertiary Template-Directed Insertions during Nonhomologous Recombination

“…The polyadenylation cassette used in humanTM is subject to up to 20% readthrough (3), thus suggesting that as many as onethird of all humanTM RIO vector-containing virions may have contained readthrough sequences. Sequences housed on readthrough RNAs are known to be packageable and to serve readily as "patch repair" recombination templates (3,20,37,53,56,69).…”