EFFICIENT INITIATION AND STRAND TRANSFER OF POLYPURINE TRACT-PRIMED PLUS-STRAND DNA PREVENT STRAND TRANSFER OF INTERNALLY INITIATED PLUS-STRAND DNAS

A critical step in retroviral reverse transcription is the initiation of plus-strand DNA synthesis at the polypurine tract (PPT) and strand transfer of the PPT-primed strong-stop DNA to the 5' end of the viral DNA. An attachment site (aft) immediately 3' to the PPT is essential f or proper integration of proviral DNA into the host chromosome. Plus-s trand DNA synthesis is discontinuous in many retroviruses, indicating that sequences upstream of the PPT are also used to initiate plus-stra nd DNA synthesis (internally initiated DNA). Strand transfer of intern ally initiated DNA would result in ''dead'' viral DNA that lacks the a tt site needed for integration. Strand transfer of the internally init iated DNA could occur if DNA synthesis failed to initiate at the PPT o r if the PPT-primed DNA was displaced before strand transfer. We sough t to determine the efficiency of DNA synthesis initiation at the PPT a nd the proportions of PPT-primed DNA and internally initiated DNAs tha t are utilized for strand transfer. We constructed spleen necrosis vir us-based retroviral vectors containing an internal PPT and an art site 5' of the normal PPT and att site. After one replication cycle of the retroviral vectors, the structures of the resulting proviruses were d etermined by Southern blotting. The analysis suggested that the PPT is an efficient and rapid initiator of plus-strand DNA synthesis and tha t internally initiated DNAs are rarely utilized for strand transfer. W e hypothesize that efficient synthesis and strand transfer of PPT-prim ed DNA evolved to prevent lethal strand transfers of internally initia ted DNAs.