Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

The nucleocapsid (NC) protein of human immunodeficiency virus HIV-1 (NCp7) is responsible for packaging the viral RNA by recognizing a packaging site (PSI) on the viral RNA genome. NCp7 is a molecule of 55 amino acids containing two zinc fingers, with only the first one being highly conserved among retroviruses. The first zinc finger is flanked by two basic amino acid clusters. Here we demonstrate that chemically synthesized NCp7 specifically binds to viral RNA containing the PSI using competitive filter binding assays. Deletion of the PSI from the RNA abrogates this effect. The 35 N-terminal amino acids of NCp7, comprising the first zinc finger, are sufficient for specific RNA binding. Chemically synthesized mutants of the first zinc finger demonstrate that the amino acid residues C-C-C/H-C/H are required for specific RNA binding and zinc coordination. Amino acid residues F16 and T24, but not K20, E21 and G22, located within this zinc finger, are essential for specific RNA binding as well. The second zinc finger cannot replace the first one. Furthermore, mutations in the basic amino acid residues flanking the first zinc finger demonstrate that R3, 7, 10, 29 and 32 but not K11, 14, 33 and 34 are also essential for specific binding. Specific binding to viral RNA is also observed with recombinant NCp15 and Pr55Gag. The results demonstrate for the first time specific interaction of a retroviral NC protein with its PSI RNA in vitro.     

Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent.

The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions. In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions. In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination. These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity. However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10. These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly. Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions.     

Structure of the His44 --> Ala single point mutant of the distal finger motif of HIV-1 nucleocapsid protein: a combined NMR, molecular dynamics simulation, and fluorescence study

The nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers that strongly bind Zn(2+) through coordination of one His and three Cys residues. It has been suggested that NCp7 function is conformation specific since substitution of any of the zinc coordinating residues in the zinc finger motifs leads to subsequent loss of viral infectivity. To further determine the structural requirements necessary for this specific conformation, we investigated by (1)H 2D NMR and molecular dynamics simulations the structure of the distal finger motif of NCp7 in which the zinc coordinating amino acid, His 44, was substituted by a noncoordinating Ala residue. While the fold of the N-terminal part of this mutated peptide was similar to that of the native peptide, an increased lability and significant conformational changes were observed in the vicinity of the His-to-Ala mutation. Moreover, molecular dynamics simulations suggested a mechanism by which the variant peptide can bind zinc ion even though one zinc-coordinating amino acid was lacking. Using the fluorescence of the naturally occurring Trp37 residue, the binding affinity of the variant peptide to the (TG)(3) model oligonucleotide was found to be decreased by about 2 orders of magnitude with respect with the native peptide. Modeling of the DNA:NCp7 complex using structures of the variant peptide suggests that the residues forming a hydrophobic cleft in the native protein are improperly oriented for efficient DNA binding by the variant peptide.     

NMR structure of the complex between the zinc finger protein NCp10 of Moloney murine leukemia virus and the single-stranded pentanucleotide d(ACGCC): comparison with HIV-NCp7 complexes.

The structure of the 56 amino acid nucleocapsid protein NCp10 of retrovirus MoMuLV, which contains a single CX(2)CX(4)HX(4)C-type zinc finger, has been determined previously by NMR. The important role of NCp10 (or NCp7 for HIV-1) in the retroviral life cycle seems mainly related to their preferential binding to single-stranded nucleic acids. We report here the structure of the complex formed between the biologically active (14-53)NCp10 and the oligonucleotide d(ACGCC) in aqueous solution determined by 2D (1)H NMR based methods. The aromatic residue Trp(35) of NCp10 directs nucleic acid complexation as shown by its complete fluorescence quenching upon addition of d(ACGCC). (1)H and (31)P NMR studies support the insertion of Trp(35) between the G(3) and C(4) bases. A total of 577 NOE distance restraints, of which 40 were intermolecular, were used for the structure determination. The zinc finger provides a well-defined surface for the binding of d(ACGCC) through hydrophobic interactions and tryptophan stacking on the guanine. This latter interaction was also observed in the NMR-derived structures of the complexes between NCp7, which contains two successive zinc fingers, and single-stranded DNA and RNA, supporting the proposal for a major role played by aromatic residues of NCp proteins in nucleic acid recognition. Upon binding to the nucleotide a new loop in NCp10 that participates in the intermolecular interaction is formed. Additional interactions provided by positively charged residues surrounding the zinc finger appear necessary for tight binding. The structure of the complex NCp10-d(ACGCC) gives a structural explanation for the loss of virus infectivity following point mutations in the finger domain.     

RNA recognition mechanism of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein

NCp8 of HIV-2 contains two CCHC-type zinc fingers connected by a linker, and is involved in many critical steps of the virus life cycle. It was previously shown that the first zinc finger flanked by the linker is the minimal active domain for specific binding to viral RNA. In our previous study, we determined the three-dimensional structure of NCp8-f1, including the minimal active domain, and found that a hydrogen bond between Asn(11) N(delta)H and Arg(27) O stabilized the conformation of the linker in the vicinity of the zinc finger [Kodera et al. (1998) Biochemistry 37, 17704-17713]. In this study, RNA binding activities of NCp8-f1 and three types of its mutant peptides were analysed by native PAGE assay. The activity and three-dimensional structure of NCp8-f1/N11A, in which alanine is substituted for Asn(11) thereby affecting the conformation of the linker, was analyzed and compared with those of NCp8-f1. We demonstrated that the existence of Arg(4) and/or Lys(5) and Arg(26) and/or Arg(27) were necessary for binding RNA. Furthermore, the linker's flexible orientation, which is controlled by the hydrogen bond between Asn(11) N(delta)H and Arg(27) O, appears to be a structural basis for NCp8 existing as a multi-functional protein.     

Effects of temperature on the dynamic behaviour of the HIV-1 nucleocapsid NCp7 and its DNA complex

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers and is involved in many crucial steps of the virus life-cycle. A large number of physiological r?les of NCp7 involve its binding to single-stranded nucleic acid chains. Several solution structures of NCp7 and its complex with single-stranded RNA or DNA have been reported. We have investigated the changes in the dynamic behaviour experienced by the (12-53)NCp7 peptide upon DNA binding using (15)N heteronuclear relaxation measurements at 293 K and 308 K, and fluorescence spectroscopy. The relaxation data were interpreted using the reduced spectral density approach, which allowed the high-frequency motion, overall tumbling rates and the conformational exchange contributions to be characterized for various states of the peptide without using a specific motional model. Analysis of the temperature-dependent correlation times derived from both NMR and fluorescence data indicated a co-operative change of the molecular shape of apo (12-53)NCp7 around 303 K, leading to an increased hydrodynamic radius at higher temperatures. The binding of (12-53)NCp7 to a single-stranded d(ACGCC) pentanucleotide DNA led to a reduction of the conformational flexibility that characterized the apo peptide. Translational diffusion experiments as well as rotational correlation times indicated that the (12-53)NCp7/d(ACGCC) complex tumbles as a rigid object. The amplitudes of high-frequency motions were restrained in the complex and the occurrence of conformational exchange was displaced from the second zinc finger to the linker residue Ala30.     

Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G approximately I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.     

Solid phase synthesis of the retroviral nucleocapsid protein NCp10 of Moloney murine leukaemia virus and related "zinc-fingers" in free SH forms. Influence of zinc chelation on structural and biochemical properties.

The core of retroviruses contains a highly conserved, low molecular weight, basic protein that binds nucleic acids and is essential for genomic RNA packaging. The 56 amino acid protein, NCp10, of Moloney Murine Leukaemia virus (MoMuLV) has the CysX2 CysX4 HisX4 Cys zinc finger-like motif shared by all retrovirus nucleocapsid proteins. The native protein and five modified peptides containing the zinc binding domain were synthesized by solid phase in order to investigate the structural and biochemical role of Zn2+ chelation in MoMuLV NCp10 activity. The purity of the synthetic molecules was verified by HPLC and their sequences were confirmed by amino acid analysis and sequencing in the case of NCp10. Thiol dosage agreed with the theoretical value of free cysteine for all these molecules. Fluorescence measurements performed on synthetic NCp10 and zinc finger fragments showed that the tryptophan quantum yield was Zn2(+)-dependent, allowing a 1:1 stoichiometry for the complex to be determined. The apparent affinity constant of NCp10 for the metal was estimated to be superior to 10(6) M-1. The synthetic protein, in the presence of Zn2+ ions, possesses all the biological properties of NCp10 isolated from virions. It catalyzes both the MoMuLV RNA dimerization and the annealing of the replication primer tRNA(Pro) onto MoMuLV RNA.     

G-quartets direct assembly of HIV-1 nucleocapsid protein along single-stranded DNA

The d(TTGGGGGGTACAGTGCA) sequence, derived from the human immunodeficiency virus type 1 (HIV-1) central DNA flap, can form in vitro an intermolecular parallel DNA quadruplex. This work demonstrates that the HIV-1 nucleocapsid protein (NCp) exhibits a high affinity (10⁸ M^–1) for this quadruplex. This interaction is predominantly hydrophobic, maintained by a stabilization between G-quartet planes and the C-terminal zinc finger of the protein. It also requires 5 nt long tails flanking the quartets plus both the second zinc-finger and the N-terminal domain of NCp. The initial binding nucleates an ordered arrangement of consecutive NCp along the four single-stranded tails. Such a process requires the N-terminal zinc finger, and was found to occur for DNA site sizes shorter than usual in a sequence-dependent manner. Concurrently, NCp binding is efficient on a G′2 quadruplex also derived from the HIV-1 central DNA flap. Apart from their implication within the DNA flap, these data lead to a model for the nucleic acid architecture within the viral nucleocapsid, where adjacent single-stranded tails and NCp promote a compact assembly of NCp and nucleic acid growing from stably and primary bound NCp.     

DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection

The nucleocapsid (NC) protein NCp7 of the immunodeficiency virus type 1 is a small basic protein with two zinc finger motifs. NCp7 has key roles in virus replication and structure, which rely on its interactions with nucleic acids. Although most interactions involve RNAs, binding to the viral DNA is thought to be of importance to achieve protection of the DNA against cellular nucleases and its integration into the host genome. We investigated the interaction of NCp7 with plasmid DNA as a model system. The fluorescence probe YOYO-1 was used as the reporter. Binding of NCp7 to DNA caused DNA condensation, as inferred from the dramatic decrease in YOYO-1 fluorescence. Efficient condensation of DNA required the full length NCp7 with the zinc fingers. The fingerless peptide was less efficient in condensing DNA. Binding of both these NC peptides led to freezing of the segmental dynamics of DNA as revealed by anisotropy decay kinetics of YOYO-1. The truncated peptide NC(12–55) which retains the zinc fingers did not lead to DNA condensation despite its ability to bind and partially freeze the segmental motion of DNA. We propose that the histone-like property of NCp7 leading to DNA condensation contributes to viral DNA stability, in vivo.