title: Characterization of (+) strand initiation and termination sequences located at the center of the equine infectious anemia virus genome.
abstract: 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.
authors: Department of Biochemistry, Division of Infectious Diseases, and Center for AIDS Research, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4984, USA.
authors: S R
authors: SR
authors: Stetor
authors: J W
authors: JW
authors: Rausch
authors: M J
authors: MJ
authors: Guo
authors: J P
authors: JP
authors: Burnham
authors: L R
authors: LR
authors: Boone
authors: M J
authors: MJ
authors: Waring
authors: S F
authors: SF
authors: Le Grice
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D016679:Genome, Viral; D006736:Horses; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral