Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

• Klíčová slova
• HIV-1, RNA packaging, fullerene, inhibition, nucleocapsid,
• MeSH
• fullereny metabolismus   farmakologie   MeSH
• genom virový účinky léků   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HEK293 buňky MeSH
• HIV-1 účinky léků   genetika   metabolismus   fyziologie   MeSH
• látky proti HIV metabolismus   farmakologie   MeSH
• lidé MeSH
• nukleokapsida - proteiny metabolismus   MeSH
• reverzní transkripce MeSH
• RNA virová metabolismus   MeSH
• svlékání virového obalu účinky léků   MeSH
• vazba proteinů MeSH
• virion metabolismus   MeSH
• zabalení virového genomu účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fullereny MeSH
• genové produkty gag - virus lidské imunodeficience MeSH
• látky proti HIV MeSH
• nukleokapsida - proteiny MeSH
• RNA virová MeSH

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein-protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

• Klíčová slova
• HIV-1 CA inhibitor, PF74 derivatives, disassembly, uncoating,
• MeSH
• HIV-1 účinky léků   MeSH
• indoly chemická syntéza   chemie   farmakologie   MeSH
• látky proti HIV chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• racionální návrh léčiv MeSH
• rekombinantní proteiny MeSH
• sestavení viru účinky léků   MeSH
• techniky syntetické chemie MeSH
• virion účinky léků   ultrastruktura   MeSH
• virové plášťové proteiny antagonisté a inhibitory   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indoly MeSH
• látky proti HIV MeSH
• rekombinantní proteiny MeSH
• virové plášťové proteiny MeSH

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

• MeSH
• HIV infekce farmakoterapie   MeSH
• HIV-1 účinky léků   fyziologie   MeSH
• látky proti HIV chemie   izolace a purifikace   farmakologie   MeSH
• lidé MeSH
• nukleokapsida analýza   účinky léků   MeSH
• proteiny virového jádra chemie   genetika   metabolismus   MeSH
• RNA virová genetika   MeSH
• rychlé screeningové testy MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• svlékání virového obalu účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• látky proti HIV MeSH
• proteiny virového jádra MeSH
• RNA virová MeSH

Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein-protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.

• Klíčová slova
• Cysteine mutagenesis, M-PMV capsid, M-PMV infectivity, Retrovirus assembly, Virus core stability,
• MeSH
• buněčné linie MeSH
• cystein genetika   MeSH
• genetické vektory MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   fyziologie   MeSH
• mutace MeSH
• proviry genetika   MeSH
• sestavení viru * MeSH
• virion fyziologie   MeSH
• virové plášťové proteiny chemie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cystein MeSH
• virové plášťové proteiny MeSH

BACKGROUND: Formation of a mature core is a crucial event for infectivity of retroviruses such as Mason-Pfizer monkey virus (M-PMV). The process is triggered by proteolytic cleavage of the polyprotein precursor Gag, which releases matrix, capsid (CA), and nucleocapsid proteins. Once released, CA assembles to form a mature core - a hexameric lattice protein shell that protects retroviral genomic RNA. Subtle conformational changes within CA induce the transition from the immature lattice to the mature lattice. Upon release from the precursor, the initially unstructured N-terminus of CA is refolded to form a β-hairpin stabilized by a salt bridge between the N-terminal proline and conserved aspartate. Although the crucial role of the β-hairpin in the mature core assembly has been confirmed, its precise structural function remains poorly understood. RESULTS: Based on a previous NMR analysis of the N-terminal part of M-PMV CA, which suggested the role of additional interactions besides the proline-aspartate salt bridge in stabilization of the β-hairpin, we introduced a series of mutations into the CA sequence. The effect of the mutations on virus assembly and infectivity was analyzed. In addition, the structural consequences of selected mutations were determined by NMR spectroscopy. We identified a network of interactions critical for proper formation of the M-PMV core. This network involves residue R14, located in the N-terminal β-hairpin; residue W52 in the loop connecting helices 2 and 3; and residues Q113, Q115, and Y116 in helix 5. CONCLUSION: Combining functional and structural analyses, we identified a network of supportive interactions that stabilize the β-hairpin in mature M-PMV CA.

• MeSH
• AIDS opičí genetika   metabolismus   MeSH
• buněčné linie MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• sekundární struktura proteinů genetika   MeSH
• sekvence aminokyselin MeSH
• sestavení viru genetika   MeSH
• virion genetika   metabolismus   MeSH
• virové plášťové proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH

Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a beta-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the beta-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA.

• MeSH
• bodová mutace * MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• sestavení viru genetika   fyziologie   MeSH
• virion genetika   metabolismus   MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH