HIV-1 Nucleocapsid Protein Research Articles

Cationic Residues of the HIV-1 Nucleocapsid Protein Enable DNA Condensation to Maintain Viral Core Particle Stability during Reverse Transcription.

The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC's basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC's high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC's basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects.

HIV-1 nucleocapsid protein binds double-stranded DNA in multiple modes to regulate compaction and capsid uncoating.

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to observe the activity of basic residue mutations of NC. All basic residue mutations lead to a defective interaction with the DNA substrate. Notably, the constant force “plateau” observed with wild-type NC is missing or largely diminished. However, the NC mutants that are still able to compact dsDNA, at least to some extent, create a DNA condensate that persists even at high forces in our stretching experiments. We investigate the nature of this high kinetic barrier for DNA decompaction, which may come from either direct NC-induced DNA crosslinking or, alternatively, from the steric entanglement of DNA in its compacted state. In either case, the slow kinetics of DNA decompaction from its NC-condensed state is likely responsible for the few hours delay between the completion of HIV-1 DNA synthesis inside the mature capsid, and subsequent capsid breaking, i.e. uncoating.

Structural basis of sequence-specific RNA recognition by the antiviral factor APOBEC3G

An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the mechanism of APOBEC3G capturing viral RNA is unknown. Here, we report crystal structures of a primate APOBEC3G bound to different types of RNAs, revealing that APOBEC3G specifically recognizes unpaired 5’-AA-3’ dinucleotides, and to a lesser extent, 5’-GA-3’ dinucleotides. APOBEC3G binds to the common 3’A in the AA/GA motifs using an aromatic/hydrophobic pocket in the non-catalytic domain. It binds to the 5’A or 5’G in the AA/GA motifs using an aromatic/hydrophobic groove conformed between the non-catalytic and catalytic domains. APOBEC3G RNA binding property is distinct from that of the HIV nucleocapsid protein recognizing unpaired guanosines. Our findings suggest that the sequence-specific RNA recognition is critical for APOBEC3G virion packaging and restricting HIV infectivity.

Mercapto-Benzamide Inhibitors effects on HIV NCp7 Protein: a parameter-free DFT based structural study

The action of the Mercapto-Benzamide (MB) class of molecules on the HIV Nucleocapsid protein (HIV NCp7), a zinc finger protein, is an issue of relatively recent research interest, relevant to develop a new class of effective and well tolerated HIV antivirals, able to overcome virus escape strategies. MB molecules are easily and cheaply synthesized, and show the ability to unfold the HIV Zinc-finger region, thus avoiding effective viral replication. This effect is not still fully understood, and moreover is highly influenced by the precise composition of MB aromatic ring and chain. Our approach to this biological problem is to adopt a quantum parameter-free (ab-initio: AI) geometrical scheme based on density functional theory (DFT) for the treatment of the electronic degrees of freedom to study with atomistic resolution the action mechanism of MB molecules on NCp7. In particular with respect to the role played by each MB functional group.We report and discuss the outcomes of the here proposed DFT simulations with respect to the different final configurational structures obtained.

Multifaceted Aspects of HIV-1 Nucleocapsid Inhibition by TAR-Targeting Peptidyl-Anthraquinones Bearing Terminal Aromatic Moieties.

2,6-dipeptidyl-anthraquinones are polycyclic planar systems substituted at opposite ring positions by short aminoacyl side chains. Derivatives with positively charged terminal amino acids showed in vitro inhibition of HIV-1 nucleocapsid (NC) protein correlating with threading intercalation through nucleic acid substrates. We found that the variation of the terminal amino acid into an aromatic moiety has profound effects on the NC inhibition of TAR–RNA melting, granting enhanced interaction with the protein. While all compounds showed appreciable NC and TAR binding, they exhibited different strengths driven by the length of the peptidyl side chains and by the stereochemistry of the terminal tyrosine. Unexpectedly, the best inhibitors of NC-induced TAR melting, characterized by the D- configuration of tyrosine, were able to form ternary complexes without competing with TAR–NC recognition sites, as shown by native mass spectrometry experiments. Furthermore, the hydrophobicity of the terminal residue enhances membrane permeation, with positive implications for further studies on these NC–TAR-targeted compounds.

HIV-1 nucleocapsid protein binds double stranded DNA in multiple modes for maximum density compaction

The nucleocapsid protein of HIV-1 (NC) is a multi-functional protein necessary for viral replication. NC binds nucleic acids with high affinity and acts as a chaperone during reverse transcription. Recent studies have demonstrated reverse transcription occurs inside the fully intact capsid and that the timing of reverse transcription and uncoating are correlated. How exactly a nearly 10 kbp viral dsDNA genome is stably contained within a narrow capsid with diameter approximately equal to the persistence length of dsDNA, and NC's role in this process, are not well understood.

HIV-1 Nucleocapsid Protein Binds Double-Stranded DNA in Multiple Modes to Regulate Compaction and Capsid Uncoating.

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. In this study, we use optical tweezers, fluorescence imaging, and atomic force microscopy to observe NC binding a single long DNA substrate in multiple modes. We find that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. When NC is removed from solution, the globule dissipates over time, but specifically-bound NC maintains long-range DNA looping that is less compact but highly stable. Both binding modes are additionally observed using AFM imaging. These results suggest multiple binding modes of NC compact DNA into a conformation compatible with reverse transcription, regulating the genomic pressure on the capsid and preventing premature uncoating.

In Vitro Evaluation of Bis-3-Chloropiperidines as RNA Modulators Targeting TAR and TAR-Protein Interaction.

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its deserved role in the next generation of pharmaceutical R&D. We have recently probed the trans-activation response (TAR) element, an RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabilizing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analytical approaches for the study of multicomponent RNA-based interactions.

Investigation of the effect of Staufen1 overexpression on the HIV-1 virus production

In this study, we investigated how Staufen1 influences the HIV-1 production. The overexpression of Staufen1 increased virus production without any negative affect on the viral infectivity. This increase was not caused by transcriptional activation; but by influencing post-transcriptional steps. Using multiple Gag protein derivatives, we confirmed that the zinc-finger domains of the HIV-1 nucleocapsid (NC) are important for its interaction with Staufen1. We also found that Staufen1 colocalized in stress granules with the mature form of the HIV-1 NC protein.

Bis-3-Chloropiperidines Targeting TAR RNA as A Novel Strategy to Impair the HIV-1 Nucleocapsid Protein.

Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem–bulge–loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs’ recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro.

Mechanism of HIV-1 NC Protein-Induced Condensation of Double Stranded DNA as a Model for DNA Compaction during Reverse Transcription

Genomic RNA is packaged into the HIV-1 virion and reverse transcribed within the mature capsid into double stranded proviral DNA (vDNA) in the presence of ∼10 millimolar HIV-1 nucleocapsid protein (NC). NC is a small, highly basic protein known to condense all nucleic acids upon saturated binding. NC-induced condensation of vDNA is critical for maintaining viral capsid integrity during reverse transcription, ensuring its completion. However, how NC condenses double stranded DNA (dsDNA) is unknown. Here, we use optical tweezers to stretch single dsDNA molecules in the presence of wild-type HIV-1 NC and observe NC-induced dsDNA condensation. Above a critical concentration of NC, reducing the extension of the stretched dsDNA molecule at a constant rate reveals stepwise changes in extension along a constant-force plateau. The quantized nature of these steps suggests winding of dsDNA into a compact toroidal globule via stepwise accumulation of dsDNA in loops. We observe changes in the condensing force and kinetics of condensation and decondensation by varying NC concentration and salts, mimicking the process that occurs during capsid uncoating and gradual NC loss. Combining the results of these stretching experiments with fluorescence imaging allows us to visualize the NC-induced formation of DNA toroids in real time. These results help us understand the state of vDNA both within the capsid and after capsid uncoating.

Nucleocapsid protein preferentially binds the stem-loop of duplex/quadruplex hybrid that unfolds the quadruplex structure

NCp7 protein binds the duplex/quadruplex hybrid structure, which decreases the thermal stability of DNA and unfolds the G-quadruplex structure. Interestingly, the duplex in the stem-loop region is the more favorable binding site of NCp7. The NCp7 binding twists the top G-tetrad, weakens hydrogen bonding and causes K+ ejection, hence disrupting the G4 structure.

Inhibitory Effect of Lithospermic Acid on the HIV-1 Nucleocapsid Protein.

The HIV-1 nucleocapsid protein (NC) is a desirable target in antiretroviral therapy due to its high conservation among HIV-1 strains, and to its multiple and crucial roles in the HIV-1 replication cycle. Natural products represent a valuable source of NC inhibitors, with the catechol group being a privileged scaffold in NC inhibition. By coupling molecular modeling with NMR spectroscopy and fluorescence-based assays, we disclosed lithospermic acid, a catechol derivative extracted from Salvia miltiorrhizza, as a potent and chemically stable non-covalent inhibitor of the NC. Being different from other catechol derivative reported so far, lithospermic acid does not undergo spontaneous oxidation in physiological conditions, thus becoming a profitable starting point for the development of efficient NC inhibitors.

Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States.

HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with nucleic acids, and p1 and p6, two unstructured regions, p6 containing the motifs to bind ALIX, the cellular ESCRT factor TSG101 and the viral protein Vpr. The processing of Gag by the viral protease subsequently liberates NCp15 (NC-p1-p6), NCp9 (NC-p1) and NCp7, NCp7 displaying the optimal chaperone activity of nucleic acids. This review focuses on the nucleic acid binding properties of the NC domain in the different maturation states during the HIV-1 viral cycle.

Discovery of potential dual-target prodrugs of HIV-1 reverse transcriptase and nucleocapsid protein 7

In the present work, we described the design, synthesis and biological evaluation of a novel series of potential dual-target prodrugs targeting the HIV-1 reverse transcriptase (RT) and nucleocapsid protein 7 (NCp7) simultaneously. Among them, the most effective compound 7c was found to inhibit HIV-1 wild-type (WT) strain at double-digit nanomolar concentration (EC50 = 42 nM) in MT-4 cells, and sub-micromole (EC50 = 0.308 μM) to inhibit HIV-1 NL4-3 strain in TZM-bl cells. This is a significant improvement over the parent drug MT. In addition, it showed moderate inhibitory potency (EC50 = 1.329 μM) against the HIV-1 K103N/Y181C double mutant strain (MT-4 cells). The metabolic stability in human plasma of compound 7c indicated that it can release the active forms of the parent drugs MT and AZT in a linear time-independent manner and turn out to be a potential prodrug.

Pan-retroviral Nucleocapsid-Mediated Phase Separation Regulates Genomic RNA Positioning and Trafficking

SUMMARYThe duality of liquid-liquid phase separation (LLPS) of cellular components into membraneless organelles defines the nucleation of both normal and disease processes including stress granule (SG) assembly. From mounting evidence of LLPS utility by viruses, we discover that HIV-1 nucleocapsid (NC) protein condenses into zinc-finger (ZnF)-dependent LLPSs that are dynamically influenced by cytosolic factors. ZnF-dependent and Zinc (Zn2+)-chelation-sensitive NC-LLPS are formed in live cells. NC-Zn2+ ejection reverses the HIV-1 blockade on SG assembly, inhibits NC-SG assembly, disrupts NC/Gag-genomic RNA (vRNA) ribonucleoprotein complexes, and causes nuclear sequestration of NC and the vRNA, inhibiting Gag expression and virus release. NC ZnF mutagenesis eliminates the HIV-1 blockade of SG assembly and repositions vRNA to SGs. We find that NC-mediated, Zn2+-coordinated phase separation is conserved among diverse retrovirus subfamilies, illustrating that this exquisitely evolved Zn2+-dependent feature of virus replication represents a critical target for pan-antiretroviral therapies.

5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein.

The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.

Reconciling NMR Structures of the HIV-1 Nucleocapsid Protein NCp7 Using Extensive Polarizable Force Field Free-Energy Simulations.

Using polarizable (AMOEBA) and nonpolarizable (CHARMM) force fields, we compare the relative free energy stability of two extreme conformations of the HIV-1 nucleocapsid protein NCp7 that had been previously experimentally advocated to prevail in solution. Using accelerated sampling techniques, we show that they differ in stability by no more than 0.75-1.9 kcal/mol depending on the reference protein sequence. While the extended form appears to be the most probable structure, both forms should thus coexist in water explaining the differing NMR findings.

(Thia)calixarenephosphonic Acids as Potent Inhibitors of the Nucleic Acid Chaperone Activity of the HIV-1 Nucleocapsid Protein with a New Binding Mode and Multitarget Antiviral Activity.

The nucleocapsid protein (NC) is a highly conserved protein that plays key roles in HIV-1 replication through its nucleic acid chaperone properties mediated by its two zinc fingers and basic residues. NC is a promising target for antiviral therapy, particularly to control viral strains resistant to currently available drugs. Since calixarenes with antiviral properties have been described, we explored the ability of calixarene hydroxymethylphosphonic or sulfonic acids to inhibit NC chaperone properties and exhibit antiviral activity. By using fluorescence-based assays, we selected four calixarenes inhibiting NC chaperone activity with submicromolar IC50 values. These compounds were further shown by mass spectrometry, isothermal titration calorimetry, and fluorescence anisotropy to bind NC with no zinc ejection and to compete with nucleic acids for the binding to NC. Molecular dynamic simulations further indicated that these compounds interact via their phosphonate or sulfonate groups with the basic surface of NC but not with the hydrophobic plateau at the top of the folded fingers. Cellular studies showed that the most soluble compound CIP201 inhibited the infectivity of wild-type and drug-resistant HIV-1 strains at low micromolar concentrations, primarily targeting the early steps of HIV-1 replication. Moreover, CIP201 was also found to inhibit the flipping and polymerization activity of reverse transcriptase. Calixarenes thus form a class of noncovalent NC inhibitors, endowed with a new binding mode and multitarget antiviral activity.

A Class of Potent Inhibitors of the HIV-1 Nucleocapsid Protein Based on Aminopyrrolic Scaffolds

The HIV-1 nucleocapsid protein 7 (NC) is a potential target for effective antiretroviral therapy due to its central role in virus replication, mainly linked to nucleic acid (NA) chaperone activity, and low susceptibility to drug resistance. By screening a compounds library, we identified the aminopyrrolic compound CN14_17, a known carbohydrate binding agent, that inhibits the NC chaperone activity in the low micromolar range. Different from most of available NC inhibitors, CN14_17 fully prevents the NC-induced annealing of complementary NA sequences. Using fluorescence assays and isothermal titration calorimetry, we found that CN14_17 competes with NC for the binding to NAs, preferentially targeting single-stranded sequences. Molecular dynamics simulations confirmed that binding to cTAR occurs preferably within the guanosine-rich single stranded sequence. Finally, CN14_17 exhibited antiretroviral activity in the low micromolar range, although with a moderate therapeutic index. Overall, CN14_17 might be the progenitor of a new promising class of NC inhibitors.