Role of the C terminus of foamy virus Gag in RNA packaging and Pol expression

Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol proteins are expressed independently of one another, and both proteins undergo single cleavage events. Thus, the mature FV Gag protein does not consist of the matrix, capsid, and nucleocapsid (NC) proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol protein.     

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.     

Unfolding of DNA quadruplexes induced by HIV-1 nucleocapsid protein

The human immunodeficiency virus type 1 nucleocapsid protein (NC) is a nucleic acid chaperone that catalyzes the rearrangement of nucleic acids into their thermodynamically most stable structures. In the present study, a combination of optical and thermodynamic techniques were used to characterize the influence of NC on the secondary structure, thermal stability and energetics of monomolecular DNA quadruplexes formed by the sequence d(GGTTGGTGTGGTTGG) in the presence of K⁺ or Sr²⁺. Circular dichroism studies demonstrate that NC effectively unfolds the quadruplexes. Studies carried out with NC variants suggest that destabilization is mediated by the zinc fingers of NC. Calorimetric studies reveal that NC destabilization is enthalpic in origin, probably owing to unstacking of the G-quartets upon protein binding. In contrast, parallel studies performed on a related DNA duplex reveal that under conditions where NC readily destabilizes and unfolds the quadruplexes, its effect on the DNA duplex is much less pronounced. The differences in NC's ability to destabilize quadruplex versus duplex is in accordance with the higher ΔG of melting for the latter, and with the inverse correlation between nucleic acid stability and the destabilizing activity of NC.     

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single lambda-DNA molecules through the helix-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7's capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity.     

Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1.

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.     

Amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human APOBEC3G packaging

APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.     

Retroviral nucleocapsid proteins display nonequivalent levels of nucleic acid chaperone activity

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC's chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NC's highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 ~ RSV > MLV HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single- and double-stranded oligonucleotides suggested that all four NC proteins have moderate duplex destabilization capabilities. Single-molecule DNA-stretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.     

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.     

Comparative structural effects of HIV-1 Gag and nucleocapsid proteins in binding to and unwinding of the viral RNA packaging signal

The major RNA binding region of the HIV-1 Gag polyprotein is the nucleocapsid (NC) domain, which is responsible for the specific capture of the genomic RNA genome during viral assembly. The Gag polyprotein has other RNA chaperone functions, which are mirrored by the isolated NC protein after physiological cleavage from Gag. Gag, however, is suggested to have superior nucleic acid chaperone activity. Here we investigate the interaction of Gag and NC with the core RNA structure of the HIV-1 packaging signal (Ψ), using 2-aminopurine substitution to create a series of modified RNAs based on the Ψ helix loop structure. The effects of 2-aminopurine substitution on the physical and structural properties of the viral Ψ were characterized. The fluorescence properties of the 2-aminopurine substitutions showed features consistent with the native GNAR tetraloop. Dissociation constants (K(d)) of the two viral proteins, measured by fluorescence polarization (FP), were similar, and both NC and Gag affected the 2-aminopurine fluorescence of bases close to the loop binding region in a similar fashion. However, the influence of Gag on the fluorescence of the 2-aminopurine nucleotides at the base of the helix implied a much more potent helix destabilizing action on the RNA stem loop (SL) versus that seen with NC. This was further supported when the viral Ψ SL was tagged with a 5' fluorophore and 3' quencher. In the absence of any viral protein, minimal fluorescence was detected; addition of NC yielded a slight increase in fluorescence, while addition of the Gag protein yielded a large change in fluorescence, further suggesting that, compared to NC, the Gag protein has a greater propensity to affect RNA structure and that Ψ helix unwinding may be an intrinsic step in RNA encapsidation.