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.

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.…”

The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors

“…54 The transfer plasmid was reassembled, retaining the left and right LTRs. The extended packaging signal (from nucleotide position 287-667, using as reference, GenBank accession number AF028232) was inserted after the left LTR, followed by a track of DNA containing the central polypurine tract/central termination sequence (nucleotide position 4812-5008) 55 and the Rev response element (nucleotide position 5400-5531). 54 All Woodchuck hepatitis virus sequences were removed from the provided transfer vector and replaced with the modified Woodchuck hepatitis virus post-transcriptional regulatory element.…”

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy

“…Gene transfer to respiratory musculature in utero LG Gregory et al from EIAV (cppt) 22 and the WPRE element from the woodchuck hepatitis virus. 23 The construction of an EIAV construct expressing nuclear localizing b-galactosidase incorporating the SV40 nuclear localization signal (SMART3 NZ) was obtained by PCR amplification from the plasmid vector pNGVL1-ntbta-gal.…”