Formation of stable and functional HIV-1 nucleoprotein complexes in vitro.
HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.
Tanchou
V
V
LaboRetro, Unité de Virologie humaine (INSERM-ENS 412), Ecole Normale Supérieure, Lyon, France.
Gabus
C
C
Rogemond
V
V
Darlix
J L
JL
D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D006163:Ribonuclease T1 / Q000378:metabolism; D013698:Templates, Genetic; D014764:Viral Proteins; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D006163:Ribonuclease T1