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, 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.

Characterization of (+) strand initiation and termination sequences located at the center of the equine infectious anemia virus genome.

Permeabilized preparations of equine infectious anemia virus (EIAV) are shown here to support efficient and accurate synthesis of full-length double-stranded proviral DNA. When (-) and (+) strand products were analyzed by Southern blotting, a discontinuity, mapping approximately to the center of the EIAV genome, could be demonstrated for the (+) strand, predicting a second site for initiation of DNA synthesis and a specific mechanism of (+) strand termination. Precise localization of this (+) strand origin within the integrase (IN) coding region was achieved through its in vitro selection and extension into, and excision from, nascent DNA by purified recombinant p66/p51 EIAV reverse transcriptase (RT), suggesting that the EIAV genome harbors a central polypurine tract (cPPT). In addition, a model system was developed for evaluating whether sequences immediately downstream of the cPPT would terminate (+) strand synthesis in the context of strand displacement. Such a sequence was indeed discovered which functions in a manner analogous to that of the central termination sequence (CTS) of HIV, where A-tract-induced minor groove compression has been suggested to induce localized distortion of the nucleic acid duplex and termination of (+) strand synthesis. This interpretation is reinforced by experiments indicating that read-through of the CTS can be efficiently promoted by substituting 2,6-diaminopurine for adenine, thereby relieving minor groove compression. The nucleotide substitution can also shift the site of termination in strand displacement (+) strand synthesis. Collectively, our data support proposals that lentiviruses may have evolved specialized mechanisms for initiating and terminating (+) strand DNA synthesis at the center of their genomes.

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.