Molecular mechanism of sequence-specific termination of lentiviral replication.

Abstract

The central termination sequence (CTS) terminates (+) strand DNA synthesis in certain lentiviruses. The molecular mechanism underlying this event, catalyzed by equine infectious anemia virus reverse transcriptase (EIAV RT), was evaluated by pre-steady-state kinetic techniques. Time courses in nucleotide incorporation using several DNA substrates were biphasic, consistent with release of enzyme from extended DNA being the rate-limiting step for turnover. While the burst amplitude reflecting the amount of functional RT-DNA complex was sequence-dependent, rate constants for initial product formation were not. Filter binding assays indicate the K(d) for CTS-containing substrate is only 2-fold higher than a random DNA and cannot account entirely for the large diminution in burst amplitudes. Measurements of processive DNA replication on a millisecond time scale indicate that the rate of polymerization is unaffected by the T(6)-tract within the CTS. However, termination products accumulate due to a substantial increase in the rate of nonproductive enzyme-nucleic acid complex formation after incorporation of four to five adenosines of a T(6)-tract within the CTS. During strand displacement synthesis through the CTS, products accumulate after incorporation of three to four adenosines. The rate of polymerization during strand displacement synthesis decreases 2-fold while the rate of nonproductive enzyme-nucleic acid complex formation is identical in the absence or presence of the displacement strand. These results have allowed us to develop a model for CTS-induced termination of (+) strand synthesis.