Identification of a central DNA flap in feline immunodeficiency virus.
A duplication of the polypurine tract (PPT) at the center of the human immunodeficiency virus type 1 (HIV-1) genome (the cPPT) has been shown to prime a separate plus-strand initiation and to result in a plus-strand displacement (DNA flap) that plays a role in nuclear import of the viral preintegration complex. Feline immunodeficiency virus (FIV) is a lentivirus that infects nondividing cells, causes progressive CD4(+) T-cell depletion, and has been used as a substrate for lentiviral vectors. However, the PPT sequence is not duplicated elsewhere in the FIV genome and a central plus-strand initiation or strand displacement has not been identified. Using Southern blotting of S1 nuclease-digested FIV preintegration complexes isolated from infected cells, we detected a single-strand discontinuity at the approximate center of the reverse-transcribed genome. Primer extension analyses assigned the gap to the plus strand, and mapped the 5' terminus of the downstream (D+) segment to a guanine residue in a purine-rich tract in pol (AAAAGAAGAGGTAGGA). RACE experiments then mapped the 3' terminus of the upstream plus (U+)-strand segment to a T nucleotide located 88 nucleotides downstream of the D+ strand 5' terminus, thereby identifying the extent of D+ strand displacement and the central termination sequence of this virus. Unlike HIV, the FIV cPPT is significantly divergent in sequence from its 3' counterpart (AAAAAAGAAAAAAGGGTGG) and contains one and in some cases two pyrimidines. An invariant thymidine located -2 to the D+ strand origin is neither required nor optimal for codon usage at this position. Although the mapped cPPTs of FIV and HIV-1 act in cis, they encode homologous amino acids in integrase.
Whitwam
T
T
Molecular Medicine Program and Division of Infectious Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.
Peretz
M
M
Poeschla
E
E
D000818:Animals; D001483:Base Sequence; D002415:Cats; D004279:DNA, Viral / Q000235:genetics*; D016679:Genome, Viral; D006801:Humans; D016089:Immunodeficiency Virus, Feline / Q000235:genetics*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral