Relative Rates of Retroviral Reverse Transcriptase Template Switching during RNA- and DNA-Dependent DNA Synthesis

Abstract

ABSTRACT Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 400-bp direct repeat. The directly repeated sequences were upstream of the polypurine tract (PPT) in the RB-LLP vector; the same direct repeats flanked the PPT and attachment site ( att ) in the RB-LPL vector. RT template switching events could occur during either RNA- or DNA-dependent DNA synthesis and delete one copy of the direct repeat plus the intervening sequences. RB-LLP vectors that underwent direct repeat deletions during RNA- and DNA-dependent DNA synthesis generated viral DNA that could integrate into the host genome. However, any deletion of the direct repeats in the RB-LPL vector that occurred during RNA-dependent DNA synthesis resulted in deletion of the essential PPT and att site and generated a dead-end viral DNA product. Thus, only RB-LPL vectors that underwent direct repeat deletions during DNA-dependent DNA synthesis could integrate to form proviruses. The RB-LLP and RB-LPL vectors were permitted to undergo a single replication cycle, and the frequencies of direct repeat deletions were determined by PCR and Southern analysis of the resulting proviruses. A comparison of the frequency of direct repeat deletions in the RB-LLP and RB-LPL vectors indicated that the in vivo rates of RT template switching during RNA- and DNA-dependent DNA synthesis are nearly identical.