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.

FIV: from lentivirus to lentivector

“…A short region of downstream (D+) strand displacement (overlap) also occurs when upstream (U+) strand synthesis terminates at the central termination sequence (CTS) approximately 100 nt beyond the origin of the HIV-1 D+ strand [118]. Similar cPPT/CTS elements have been identified in studies of equine infectious anemia virus (EIAV) templates [119,120]. The resulting overlap of 88-98 nt in the HIV-1 pre-integration complex has been termed the central DNA flap [118].…”

“…In contrast to the CTS, the intra-flap 88 nt preceding the FIV CTS displays little homology to the analogous 88 nt in HIV-1. Two similar motifs have also been found to act as strong pause determinants for purified EIAV reverse transcriptase on in vitro templates [119,120]. Given the conflicting data about the function of the flap in HIV-1 [104,[123][124][125][126], it will be of interest to determine the functional role in FIV.…”

Kinetics of Association and Dissociation of HIV-1 Reverse Transcriptase Subunits

“…The 25/36-mer random sequence DNA described previously was used as substrate (18). The oligonucleotides were purified; the duplex was annealed, purified, and 5’-end labeled with [γ- 33 P]ATP (19).…”

Evaluating the Effects of Enhanced Processivity and Metal Ions on Translesion DNA Replication Catalyzed by the Bacteriophage T4 DNA Polymerase

“…Indeed, we have reported previously that the combined effects of strand displacement DNA synthesis upon encountering poly(A) tracts of nucleic acid facilitates the termination of DNA synthesis catalyzed by the human immunodeficiency virus I reverse transcriptase. 60…”