Molecular mechanism of sequence-specific termination of lentiviral replication

The central termination sequence (CTS) terminates (+) strand DNA synthesis in certain lentiviruses. The molecular mechanism underlying this event. cat alyzed by equine infectious anemia virus reverse transcriptase (EIAV RT), w as evaluated by pre-steady-state kinetic techniques. Time courses in nucleo tide incorporation using several DNA substrates were biphasic, consistent w ith release of enzyme from extended DNA being the rate-limiting step for tu rnover. While the burst amplitude reflecting the amount of functional RT-DN A complex was sequence-dependent, rate constants for initial product format ion were not. Filter binding assays indicate the Kd for CTS-containing subs trate is only 2-fold higher than a random DNA and cannot account entirely f or the large diminution in burst amplitudes. Measurements of processive DNA replication on a millisecond time scale indicate that the rate of polymeri zation is unaffected by the T-6-tract within the CTS. However, termination products accumulate due to a substantial increase in the rate of nonproduct ive enzyme-nucleic acid complex formation after incorporation of four to fi ve adenosines of a T-6-tract within the CTS. During strand displacement syn thesis through the CTS, products accumulate after incorporation of three to four adenosines. The rate of polymerization during strand displacement syn thesis decreases 2-fold while the rate of nonproductive enzyme-nucleic acid complex formation is identical in the absence or presence of the displacem ent strand. These results have allowed us to develop a model for CTS-induce d termination of (+) strand synthesis.