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 sho wn here to support efficient and accurate synthesis of full-length double-s tranded proviral DNA. When (-) and (+) strand products were analyzed by Sou thern blotting, a discontinuity, mapping approximately to the center of the EIAV genome, could be demonstrated for the (+) strand, predicting a second site fur initiation of DNA synthesis and a specific mechanism of (+) stran d termination. Precise localization of this (+) strand origin within the in tegrase (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 harbor s a central polypurine tract (cPPT), In addition, a model system was develo ped for evaluating whether sequences immediately downstream of the cPPT wou ld terminate (+) strand synthesis in the context of strand displacement. Su ch a sequence was indeed discovered which functions in a manner analogous t o that of the central termination sequence (CTS) of HIV, where A-tract-indu ced minor groove compression has been suggested to induce localized distort ion of the nucleic acid duplex and termination of (+) strand synthesis. Thi s interpretation is reinforced by experiments indicating that read-through of the CTS can be efficiently promoted by substituting 2,6-diaminopurine fo r adenine, thereby relieving minor groove compression. The nucleotide subst itution can also shift the site of termination in strand displacement (+) s trand 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.