title: Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription.
abstract: The mature HIV-1 nucleocapsid protein (NCp7) is generated by sequential proteolytic cleavage of precursor proteins containing additional C-terminal peptides: NCp15 (NCp7-spacer peptide 2 (SP2)-p6); and NCp9 (NCp7-SP2). Here, we compare the nucleic acid chaperone activities of the three proteins, using reconstituted systems that model the annealing and elongation steps in tRNA(Lys3)-primed (-) strong-stop DNA synthesis and subsequent minus-strand transfer. The maximum levels of annealing are similar for all of the proteins, but there are important differences in their ability to facilitate reverse transcriptase (RT)-catalyzed DNA extension. Thus, at low concentrations, NCp9 has the greatest activity, but with increasing concentrations, DNA synthesis is significantly reduced. This finding reflects NCp9's strong nucleic acid binding affinity (associated with the highly basic SP2 domain) as well as its slow dissociation kinetics, which together limit the ability of RT to traverse the nucleic acid template. NCp15 has the poorest activity of the three proteins due to its acidic p6 domain. Indeed, mutants with alanine substitutions for the acidic residues in p6 have improved chaperone function. Collectively, these data can be correlated with the known biological properties of NCp9 and NCp15 mutant virions and help to explain why mature NC has evolved as the critical cofactor for efficient virus replication and long-term viral fitness.
authors: Section on Viral Gene Regulation, Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2780, USA.
authors: Tiyun
authors: T
authors: Wu
authors: AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702-1201, USA.
authors: Robert J
authors: RJ
authors: Gorelick
authors: Section on Viral Gene Regulation, Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2780, USA. Electronic address: levinju@mail.nih.gov.
authors: Judith G
authors: JG
authors: Levin
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012357:RNA, Transfer, Lys / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D048348:Reverse Transcription; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D018832:Molecular Chaperones; D019590:Nucleocapsid Proteins; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus
keywords: Assembly intermediates; HIV-1; Nucleic acid chaperone; Nucleocapsid precursor proteins; Reverse transcription; Roadblock mechanism