Fig. 4: Schematic representation of six mechanisms of aberrant strand transfer during reverse transcription.

a Reverse transcription is initiated through the interaction between human tRNA(Lys-3) (orange) and the viral primer binding site on the RNA (light blue). Minus-strand cDNA (red: prior to first-strand transfer; green: post first-strand transfer) synthesis is disrupted and first-strand transfer occurs before the 5′ end of the viral RNA is reached. Premature first-strand transfers are characterized by a deletion junction in the U5 region. b Aberrant first-strand transfer events were identified either by a deletion junction within the R region or by a deletion junction coinciding with the homopolymer G triplet that defines the 5′ end of the R region. Aberrant first-strand transfers (a, b) give rise to double-stranded DNA (dsDNA) with incomplete LTR regions. c Following successful first-strand transfer, strand transfer events during minus-strand synthesis generate dsDNA with intact LTR regions and predominantly large internal deletions. d Second-strand synthesis initiates at the polypurine tract and proceeds to transcribe the 5’ end of the plus-strand DNA (dark blue). As with premature first-strand synthesis, plus-strand synthesis can also be prematurely disrupted, giving rise to defective 5′ LTR regions. d–f Strand transfer events emerging during plus-strand synthesis result in mismatched double-stranded DNA molecules. e In contrast to premature second-strand transfer, second-strand transfer can also be delayed. In this scenario, reverse transcriptase begins transcribing fragments of tRNA(Lys-3) before switching to the minus-strand template. Although LTR regions remain intact in this scenario, the plus-strand carries an insertion that originates from the tRNA(Lys-3)-primer. f Strand transfer events during plus-strand synthesis represent an additional mechanism associated with the generation of internal deletions that, for technical reasons, could not be confirmed using the analysed dataset. All deletions are represented by dotted lines. Created in BioRender. Hardy, J. (2025) https://BioRender.com/78oz5fu.