Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.     

Molecular determinants of HIV-1 NCp7 chaperone activity in maturation of the HIV-1 dimerization initiation site

Human immunodeficiency virus genome dimerization is initiated through an RNA–RNA kissing interaction formed via the dimerization initiation site (DIS) loop sequence, which has been proposed to be converted to a more thermodynamically stable linkage by the viral p7 form of the nucleocapsid protein (NC). Here, we systematically probed the role of specific amino acids of NCp7 in its chaperone activity in the DIS conversion using 2-aminopurine (2-AP) fluorescence and nuclear magnetic resonance spectroscopy. Through comparative analysis of NCp7 mutants, the presence of positively charged residues in the N-terminus was found to be essential for both helix destabilization and strand transfer functions. It was also observed that the presence and type of the Zn finger is important for NCp7 chaperone activity, but not the order of the Zn fingers. Swapping single aromatic residues between Zn fingers had a significant effect on NCp7 activity; however, these mutants did not exhibit the same activity as mutants in which the order of the Zn fingers was changed, indicating a functional role for other flanking residues. RNA chaperone activity is further correlated with NCp7 structure and interaction with RNA through comparative analysis of nuclear magnetic resonance spectra of NCp7 variants, and complexes of these proteins with the DIS dimer.     

Nucleomimetic strategy for the inhibition of HIV-1 nucleocapsid protein NCp7 activities

We report the synthesis and biological properties of three modified dinucleotides T*G, G*T and T*T in which the natural phosphodiester linkage has been replaced by a methylene carboxamide unit. They have been designed to act as nucleomimetics of a sequence recognized by the HIV-1 nucleocapsid protein NCp7 and to inhibit this interaction.     

Small-molecule inhibition of HIV-1 Vif

The HIV-1 protein Vif, essential for in vivo viral replication, targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses and hepatitis B virus. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule-based new therapies for HIV infection or for enhancing innate immunity against viruses.     

Mechanism of zinc coordination by point-mutated structures of the distal CCHC binding motif of the HIV-1 NCp7 protein

The kinetics of Zn(2+) binding by two point-mutated forms of the HIV-1 NCp7 C-terminal zinc finger, each containing tridentate binding motif HCC [Ser49(35-50)NCp7] or CCC [Ala44(35-50)NCp7], has been studied by stopped-flow spectrofluorimetry. Both the formation and dissociation rate constants of the complexes between Zn(2+) and the two model peptides depend on pH. The results are interpreted on the basis of a multistep reaction model involving three Zn(2+) binding paths due to three deprotonated states of the coordinating motif, acting as monodentate, bidentate, and tridentate ligands. For Ser49(35-50)NCp7 around neutral pH, binding preferentially occurs via the deprotonated Cys36 in the bidentate state also involving His44. The binding rate constants for the monodentate and bidentate states are 1 x 10(6) and 3.9 x 10(7) M(-)(1) s(-)(1), respectively. For Ala44(35-50)NCp7, intermolecular Zn(2+) binding predominantly occurs via the deprotonated Cys36 in the monodentate state with a rate constant of 3.6 x 10(7) M(-)(1) s(-)(1). In both mutants, the final state of the Zn(2+) complex is reached by subsequent stepwise ligand deprotonation and intramolecular substitution of coordinated water molecules. The rate constants for the intermolecular binding paths of the bidentate and tridentate states of Ala44(35-50)NCp7 and of the tridentate state of Ser49(35-50)NCp7 are much smaller than expected according to electrostatic considerations. This is attributed to conformational constraints required to achieve proper metal coordination during folding. The dissociation of Zn(2+) from both peptides is again characterized by a multistep process and takes place fastest via the protonated Zn(2+)-bound bidentate and monodentate states, with rate constants of approximately 0.3 and approximately 10(3) s(-)(1), respectively, for Ser49(35-50)NCp7 and approximately 4 x 10(-)(3) and approximately 500 s(-)(1), respectively, for Ala44(35-50)NCp7.     

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.     

Validation of an HIV-1 inactivation protocol that is compatible with intracellular drug analysis by mass spectrometry

Mass spectrometry is a powerful tool for studying the intracellular pharmacokinetics of antiretroviral drugs. However, the biohazard of HIV-1 calls for a safety protocol for such analyses. To this end, we extracted HIV-1 producing cells with methanol or ethanol at 4 degrees C. After extraction, no viral infectivity was detected, as shown by a reduction in infectious titers of more than 6log. In addition, this protocol is compatible with the quantitative analysis of antiretroviral drugs in cell extracts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Thus, using this protocol, infectious HIV-1 is inactivated and antiretroviral drugs are extracted from cells in a single step.     

HIV-1 nucleocapsid protein NCp7 and its RNA stem loop 3 partner: rotational dynamics of spin-labeled RNA stem loop 3

The tumbling dynamics of a 20-mer HIV-1 RNA stem loop 3 spin-labeled at the 5' position were probed in the nanosecond time range. This RNA interacted with the HIV-1 nucleocapsid Zn-finger protein, 1-55 NCp7, and specialized stopped-flow EPR revealed concomitant kinetics of probe immobilization from milliseconds to seconds. RNA stem loop 3 is highly conserved in HIV, while NCp7 is critical to HIV-RNA packaging and annealing. The 5' probe did not perturb RNA melting or the NCp7/RNA interaction monitored by gel shift and fluorescence. The 5'-labeled RNA tumbled with a subnanosecond isotropic correlation time (approximately 0.60 ns at room temperature) reflecting both local viscosity-independent bond rotation of the probe and viscosity-dependent diffusion of 40-60% of the RNA. The binding of NCp7 to spin-labeled RNA stem loop 3 in a 1:1 ratio increased the spin-labeled tumbling time by about 40%. At low ionic strength with a ratio of NCp7 to RNA >or=3 (i.e., an NCp7 to nucleotide ratio or=3:1 complex also required intact Zn fingers. Stopped-flow EPR kinetics with NCP7/RNA mixed at a 4:1 ratio showed the major phase of NCp7 interaction with RNA stem loop 3 occurred within 4 ms, a second phase occurred with a time constant of approximately 30 ms, and a slower immobilization, possibly concomitant with large complex formation, proceeded over seconds. This work points the way for spin-labeling to investigate oligonucleotide-protein complexes, notably those lacking precise stoichiometry, that are requisite for viral packaging and genome fabrication.     

Metabolic regulation of sodium-calcium exchange by intracellular acyl CoAs

The sodium-calcium exchanger (NCX) is a critical mediator of calcium homeostasis. In the heart, NCX1 predominantly operates in forward mode to extrude Ca(2+); however, reverse-mode NCX1 activity during ischemia/reperfusion (IR) contributes to Ca(2+) loading and electrical and contractile dysfunction. IR injury has also been associated with altered fat metabolism and accumulation of long-chain acyl CoA esters. Here, we show that acyl CoAs are novel, endogenous activators of reverse-mode NCX1 activity, exhibiting chain length and saturation dependence, with longer chain saturated acyl moieties being the most effective NCX1 activators. These results implicate dietary fat composition as a plausible determinant of IR injury. We further show that acyl CoAs may interact directly with the XIP (exchanger inhibitory peptide) sequence, a known region of anionic lipid modulation, to dynamically regulate NCX1 activity and Ca(2+) homeostasis. Additionally, our findings have broad implications for the coupling of Ca(2+) homeostasis to fat metabolism in a variety of tissues.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)     

Two basic regions of NCp7 are sufficient for conformational conversion of HIV-1 dimerization initiation site from kissing-loop dimer to extended-duplex dimer.

Nucleocapsid (NC) protein possesses nucleotide-annealing activities, which are used in various processes in retroviral life cycle. As conserved characters, the NC proteins have one or two zinc fingers of CX(2)CX(4)HX(4)C motif surrounded by basic amino acid sequences. Requirement of the zinc fingers for the annealing activities of NC protein remains controversial. In this study, we focused the requirement in the process of maturation of dimeric viral RNA. Discrimination between immature and mature dimers of synthetic RNA corresponding to the dimerization initiation site of human immunodeficiency virus type 1 (HIV-1) genomic RNA was performed based on their Mg(2+)-dependent stability in gel electrophoreses and on their distinct signal pattern from NMR analysis of imino protons. Chaperoning activity of the HIV-1 NC protein, NCp7, and its fragments for maturation of dimeric RNA was investigated using these experimental systems. We found that the two basic regions flanking the N-terminal zinc finger of NCp7, which are connected by two glycine residues instead of the zinc finger, were sufficient, although about 10 times the amounts of peptide were needed in comparison with intact NCp7. Further, it was found that the amount of basic residues rather than the amino acid sequence itself is important for the activity. The zinc fingers may involve the binding affinity and/or such a possible specific binding of NCp7 to dimerization initiation site dimer that leads to the maturation reaction.