Altering the intracellular environment increases the frequency of tandem repeat deletion during Moloney murine leukemia virus reverse transcription.

Retroviruses can persist in infected cells in the form of double-stranded DNA copies of the viral RNA genome. During reverse transcription, viral reverse transcriptase (RT) must perform two template switches—the first and second strong-stop template switches—to complete the synthesis of integration-competent viral DNA (11). It has been postulated that the requirement to perform these two obligatory template switches confers on RT the tendency to make additional, nonrequired template switches which can lead to viral genetic recombination (6, 32, 37). Paired with RT’s relatively high base substitution rate, such recombination events are thought to introduce much of the genetic variation that is observed in retroviral populations (15, 22, 30, 33). Both genetic alterations to RT and changes to the intracellular environment can affect RT-mediated base substitution rates (21, 24, 29, 38). However, factors that affect template switching during reverse transcription remain largely unknown. During recombinogenic template switching, RT begins DNA synthesis on one copy of the viral genome and then switches to the other, copackaged genome (intermolecular template switching). The frequency of recombinogenic template switching varies, but experimental evidence suggests that on average, such events happen roughly once per genome per cycle of reverse transcription (19). RT can also perform template switches between two different positions on the same template molecule (intramolecular template switching). Homologous recombination—template switching between regions with high sequence similarity—is far more frequent than recombination between nonhomologous sequences (15, 43, 44). In studies where homology patterns should permit either type of switch, intramolecular template switching was much more common than intermolecular template switching (13). Template switching during reverse transcription on templates containing direct repeats frequently results in the deletion of one copy of the repeat (3, 8, 13, 20, 22, 31, 32, 35, 36, 40). Spleen necrosis virus-based vectors with direct repeats of 1,333, 788, and 383 bp have been shown to yield deleted products 93%, 85%, and 28 to 40% of the time, respectively (20). Although recombination hot spots have been characterized and template switching frequency does not always reflect the length of sequence homology, direct-repeat deletion rates are generally roughly proportional to the length of the direct repeat (1, 19, 20, 31, 44). Early models for retroviral template switching during minus strand synthesis evoked the need for a template break to force transfer to the homologous region of the copackaged genome (6). However, the high frequency of tandem repeat deletions and the relatively minor effects on recombination of experimental conditions designed to enhance template lesions suggest that physical breaks in templates—although possibly involved in some template switching—are not required (14, 20, 40). It has also been suggested that kinetic disruptions, such as pausing by RT, may promote departure from one template and association with another. Support for this possibility comes from observations that some template features, such as RNA structures and homopolymeric runs, which may impede RT elongation, are retroviral recombination hot spots (19, 31, 39). For many types of polymerases, slowing of elongation can affect a wide range of elongation properties. For example, there is a correlation between decreased rates of elongation and pausing and/or termination for Escherichia coli RNA polymerase. An RNA polymerase mutant with decreased affinity for the substrates ATP and GTP showed a decrease in elongation rate and increased pausing (18). Additional studies in the E. coli system demonstrated that elongation rate may determine the probability of transcript release and that termination efficiency is indirectly proportional to the elongation rate (17, 25). The authors of those studies noted that small changes in the elongation rate of RNA polymerase can have large effects on termination efficiency. In this study we sought to address the effects of decreasing the RT elongation rate on template switching events, such as those that lead to retroviral genetic recombination. Previous determinations of rates of retroviral recombination have been performed in transformed cells, but viral replication in its natural setting occurs in cells whose intracellular environments are less metabolically rich than those of actively dividing cultured cells. In this study, hydroxyurea (HU) was used to produce an imbalance of intracellular nucleotide pools and template switching was scored by determining deletion rates of a 117-bp direct repeat in a lacZ reporter gene. We found that HU treatment lengthened the amount of time required to complete retroviral DNA synthesis and that template switching frequency under these conditions was increased threefold compared to that of untreated control cells.