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
Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.
- Keywords
- HIV-1, IP6, assembly, capsid, immature, mature, polyanion,
- MeSH
- gag Gene Products, Human Immunodeficiency Virus chemistry genetics metabolism MeSH
- HIV-1 chemistry genetics MeSH
- Mutation, Missense MeSH
- Polyelectrolytes MeSH
- Polymers chemistry MeSH
- Virus Assembly * MeSH
- Amino Acid Substitution MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- gag Gene Products, Human Immunodeficiency Virus MeSH
- polyanions MeSH Browser
- Polyelectrolytes MeSH
- Polymers 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
It has now been more than two years since we said our last goodbye to Jan Svoboda (14 [...].
