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
Viruses from the genus Enterovirus are important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.
- MeSH
- Cercopithecus aethiops MeSH
- dvouvláknová RNA chemie genetika MeSH
- elektronová kryomikroskopie MeSH
- enterovirus B lidský genetika ultrastruktura MeSH
- epitelové buňky ultrastruktura virologie MeSH
- genom virový * MeSH
- kapsida chemie ultrastruktura MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- RNA virová chemie genetika MeSH
- simulace molekulární dynamiky MeSH
- svlékání virového obalu genetika MeSH
- virion genetika ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH