We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

• Keywords
• DEAH-box RNA helicase, DHX15, G-patch, gRNA packaging, retrovirus,
• MeSH
• Cell Nucleus metabolism   virology   MeSH
• DEAD-box RNA Helicases metabolism   genetics   MeSH
• Genome, Viral MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus * genetics   metabolism   physiology   MeSH
• Virus Replication genetics   physiology   MeSH
• RNA, Viral * metabolism   genetics   MeSH
• RNA Helicases metabolism   genetics   MeSH
• Virus Assembly * genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DEAD-box RNA Helicases MeSH
• DHX15 protein, human MeSH Browser
• RNA, Viral * MeSH
• RNA Helicases MeSH

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.

• Keywords
• HIV-1, RNA packaging, fullerene, inhibition, nucleocapsid,
• MeSH
• Fullerenes metabolism   pharmacology   MeSH
• Genome, Viral drug effects   MeSH
• gag Gene Products, Human Immunodeficiency Virus metabolism   MeSH
• HEK293 Cells MeSH
• HIV-1 drug effects   genetics   metabolism   physiology   MeSH
• Anti-HIV Agents metabolism   pharmacology   MeSH
• Humans MeSH
• Nucleocapsid Proteins metabolism   MeSH
• Reverse Transcription MeSH
• RNA, Viral metabolism   MeSH
• Virus Uncoating drug effects   MeSH
• Protein Binding MeSH
• Virion metabolism   MeSH
• Viral Genome Packaging drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fullerenes MeSH
• gag Gene Products, Human Immunodeficiency Virus MeSH
• Anti-HIV Agents MeSH
• Nucleocapsid Proteins MeSH
• RNA, Viral MeSH

Betulinic acid (BA) is a potent triterpene, which has shown promising potential in cancer and HIV-1 treatment. Here, we report a synthesis and biological evaluation of 17 new compounds, including BODIPY labelled analogues derived from BA. The analogues terminated by amino moiety showed increased cytotoxicity (e.g., BA had on CCRF-CEM IC50 > 50 μM, amine 3 IC50 0.21 and amine 14 IC50 0.29). The cell-cycle arrest was evaluated and did not show general features for all the tested compounds. A fluorescence microscopy study of six derivatives revealed that only 4 and 6 were detected in living cells. These compounds were colocalized with the endoplasmic reticulum and mitochondria, indicating possible targets in these organelles. The study of anti-HIV-1 activity showed that 8, 10, 16, 17 and 18 have had IC50i > 10 μM. Only completely processed p24 CA was identified in the viruses formed in the presence of compounds 4 and 12. In the cases of 2, 8, 9, 10, 16, 17 and 18, we identified not fully processed p24 CA and p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat.

• Keywords
• BODIPY, betulinic acid, bevirimat, cancer, cell-cycle, cytotoxicity, fluorescent microscopy, maturation inhibitor,
• Publication type
• Journal Article 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.

• Keywords
• HIV-1 CA inhibitor, PF74 derivatives, disassembly, uncoating,
• MeSH
• HIV-1 drug effects   MeSH
• Indoles chemical synthesis   chemistry   pharmacology   MeSH
• Anti-HIV Agents chemical synthesis   chemistry   pharmacology   MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Drug Design MeSH
• Recombinant Proteins MeSH
• Virus Assembly drug effects   MeSH
• Chemistry Techniques, Synthetic MeSH
• Virion drug effects   ultrastructure   MeSH
• Capsid Proteins antagonists & inhibitors   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Indoles MeSH
• Anti-HIV Agents MeSH
• Recombinant Proteins MeSH
• Capsid Proteins 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 Infections drug therapy   MeSH
• HIV-1 drug effects   physiology   MeSH
• Anti-HIV Agents chemistry   isolation & purification   pharmacology   MeSH
• Humans MeSH
• Nucleocapsid analysis   drug effects   MeSH
• Viral Core Proteins chemistry   genetics   metabolism   MeSH
• RNA, Viral genetics   MeSH
• High-Throughput Screening Assays MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Virus Uncoating drug effects   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-HIV Agents MeSH
• Viral Core Proteins MeSH
• RNA, Viral MeSH

The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.

• Keywords
• Mason-Pfizer monkey virus, endosomes, envelope, intracellular trafficking, transport, virus-like particles,
• MeSH
• Simian Acquired Immunodeficiency Syndrome virology   MeSH
• Cell Membrane metabolism   virology   MeSH
• Endosomes metabolism   virology   MeSH
• Gene Products, env genetics   metabolism   MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Virus Assembly MeSH
• Protein Transport MeSH
• Transport Vesicles metabolism   virology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, env 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.

• Keywords
• Cysteine mutagenesis, M-PMV capsid, M-PMV infectivity, Retrovirus assembly, Virus core stability,
• MeSH
• Cell Line MeSH
• Cysteine genetics   MeSH
• Genetic Vectors MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Mutation MeSH
• Proviruses genetics   MeSH
• Virus Assembly * MeSH
• Virion physiology   MeSH
• Capsid Proteins chemistry   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cysteine MeSH
• Capsid Proteins MeSH