The HIV-1 Nef protein enhances the affinity of reverse transcriptase for RNA in vitro.
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.
Fournier
Cécile
C
Laboratoire de Virologie & Pathogénèse Virale, CNRS UMR 5537, Faculté de Médecine RTH Laennec, 7, Rue Guillaume Paradin 69008, Lyon, France.
Cortay
Jean-Claude
JC
Carbonnelle
Caroline
C
Ehresmann
Chantal
C
Marquet
Roland
R
Boulanger
Pierre
P
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
D015693:Gene Products, nef; D054311:nef Gene Products, Human Immunodeficiency Virus; D012313:RNA; D012194:RNA-Directed DNA Polymerase