A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication.
We recently reported that human immunodeficiency virus type 1 (HIV-1) unintegrated linear DNA displays a discontinuity in its plus strand, precisely defined by a second copy of the polypurine tract (PPT) located near the middle of the genome (P. Charneau and F. Clavel, J. Virol. 65:2415-2421, 1991). This central PPT appears to determine a second initiation site for retrovirus DNA plus-strand synthesis. We show here that mutations replacing purines by pyrimidines in the HIV-1 central PPT, which do not modify the overlapping amino acid sequence, are able to significantly slow down viral growth as they reduce plus-strand origin at the center of the genome. One of these mutations, introducing four pyrimidines, results in a 2-week delay in viral growth in CEM cells and abolishes plus-strand origin at the central PPT. The introduction in this mutant of a wild-type copy of the PPT at a different site creates a new plus-strand origin at that site. This new origin also determines the end of the upstream plus-strand segment, probably as a consequence of limited strand displacement-synthesis. Our findings further demonstrate the role of PPTs as initiation sites for the synthesis of the retroviral DNA plus strand and demonstrate the importance of a second such origin for efficient HIV replication in vitro.
Charneau
P
P
Département SIDA et Rétrovirus, Institut Pasteur, Paris, France.
Alizon
M
M
Clavel
F
F
D000163:Acquired Immunodeficiency Syndrome / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D015751:Genes, pol / Q000235:genetics; D015497:HIV-1 / Q000254:growth & development*; D006801:Humans; D019426:Integrases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011687:Purines; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016662:Virus Integration; D014779:Virus Replication
D004279:DNA, Viral; D011687:Purines; D004254:DNA Nucleotidyltransferases; D019426:Integrases