title: The HIV-1 Nef protein enhances the affinity of reverse transcriptase for RNA in vitro.
abstract: Several viral proteins, including nucleocapsid protein, integrase, Vif, Tat, and Nef have been proposed to act as cofactors of HIV-1 reverse transcription. Using two viral RNA probes, one overlapping the primer-binding site (PBS) and the other representing the ribosomal frameshifting signal (FS) of HIV-1 RNA, we found that recombinant full-length Nef protein (NefLAI) increased the affinity of reverse transcriptase (RT) for RNA in vitro, and interacted directly with RT in protein co-precipitation assays. The effect on RT-RNA binding and the capacity of Nef to interact with RT was also observed with N-terminal deletion mutant NefDelta57 and NefSF2, although to a lesser level. NefDelta57 corresponded to the processed Nef protein present in the internal core of mature virions, and lacked the N-myristoylated N-terminus and N-terminal region implicated in virus infectivity and pathogenicity in vivo. NefSF2, a Nef allele from a highly pathogenic strain of HIV-1, differed from NefLAI by the amino acid sequence and immunoreactivity of its N-terminal domain. The effect observed with NefSF2 and NefDelta57, and data from phage biopanning experiments suggested that the RT-binding region in Nef involved the C-terminal flexible loop of its C-terminal domain, but the function in RT-RNA binding was also influenced by its N-terminal domain.
authors.affiliation: Laboratoire de Virologie & Pathogénèse Virale, CNRS UMR 5537, Faculté de Médecine RTH Laennec, 7, Rue Guillaume Paradin 69008, Lyon, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Fournier
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Cortay
authors.forename: Caroline
authors.initials: C
authors.lastname: Carbonnelle
authors.forename: Chantal
authors.initials: C
authors.lastname: Ehresmann
authors.forename: Roland
authors.initials: R
authors.lastname: Marquet
authors.forename: Pierre
authors.initials: P
authors.lastname: Boulanger
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D015693:Gene Products, nef / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D023302:Protein Footprinting; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus
chemical_list: D015693:Gene Products, nef; D054311:nef Gene Products, Human Immunodeficiency Virus; D012313:RNA; D012194:RNA-Directed DNA Polymerase