The (52-96) C-terminal domain of Vpr stimulates HIV-1 IN-mediated homologous strand transfer of mini-viral DNA.
Viral integrase (IN) and Vpr are both components of the human immunodeficiency virus type 1 (HIV-1) pre-integration complex. To investigate whether these proteins interact within this complex, we investigated the effects of Vpr and its subdomains on IN activity in vitro. When a 21mer oligonucleotide was used as a donor and acceptor, both Vpr and its C-terminal DNA-binding domain [(52-96)Vpr] inhibited the integration reaction, whereas the (1-51)Vpr domain did not affect IN activity. Steady-state fluorescence anisotropy showed that both full-length and (52-96)Vpr bind to the short oligonucleotide, thereby extending previous observations with long DNA. The concentrations of the two proteins required to inhibit IN activity were consistent with their affinities for the oligonucleotide. The use of a 492 bp mini-viral substrate confirmed that Vpr can inhibit the IN-mediated reaction. However, the activity of (52-96)Vpr differed notably since it stimulated specifically integration events involving two homologous mini-viral DNAs. Order of addition experiments indicated that the stimulation was maximal when IN, (50-96)Vpr and the mini-viral DNA were allowed to form a complex. Furthermore, in the presence of (50-96)Vpr, the binding of IN to the mini-viral DNA was dramatically enhanced. Taken together, these data suggest that (52-96)Vpr stimulates the formation of a specific complex between IN and the mini-viral DNA.
CNRS-UMR 8532, L.B.P.A., ENS Cachan, 61 avenue du Président Wilson, 94235 Cachan, France.
Julien
J
Bischerour
Patrick
P
Tauc
Hervé
H
Leh
Hugues
H
de Rocquigny
Bernard
B
Roques
Jean-François
JF
Mouscadet
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D016336:Gene Products, vpr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011995:Recombination, Genetic; D016662:Virus Integration / Q000235:genetics; D054325:vpr Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D016336:Gene Products, vpr; D009841:Oligonucleotides; D054325:vpr Gene Products, Human Immunodeficiency Virus; D019427:HIV Integrase