A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors

Abstract

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.

PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles

“…Some experiments proved that PF74 decreased the stability of the HIV-1 core and thus accelerated its disassembly [49]. However, another paper documented that PF74 strengthened the stability of the HIV-1 CA cores, and thus slowed down the disassembly process [51,52,57]. Our data, obtained using an in vitro stabilization assay (DITH) supported the model in which PF74 acted as a stabilizer of the HIV-1 mature hexameric lattice [51].…”

“…To quantify the effect of PF74 derivatives on the stability of the viral capsid, we used a combination of in vitro, cell-based, and NMR structural assays. The data gained by our recently developed in vitro disassembly inhibitor test for HIV (DITH) [57] strongly supported the hypothesis that the mechanism of PF74 is to stabilize, not to destabilize, the mature HIV-1 CA hexameric lattice. Moreover, the higher in vitro stabilization activity of the D9 and D10 derivatives in comparison to the original PF74 pointed toward the nature of further possible modifications.…”

“…In contrast to the assembly of immature particles, PF74 was reported to affect the stability of the hexameric lattice of HIV-1 mature particles during disassembly. To quantify and compare the effects of PF74 and its derivatives on the stabilization/destabilization of the CA hexameric lattice of CANC tubular structures, we next applied the DITH assay [57]. Similarly to FAITH, this method was based on the measurement of fluorescence released from the tqON.…”

“…This assay was used as described in [57]. Tested inhibitors were added at a final concentration of 10 µM to HIV-1 CANC tubular structures in a 96-well plate, assembled as described for FAITH.…”

Antiviral immunity and nucleic acid sensing in haematopoietic stem cell gene engineering

“…Cytosolic cGAS has been shown to recognize VSV-G pseudotyped HIV-1 and other retroviral DNA in the THP-1 cell line, monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) when additional blocks to replication are removed [ 48 , 49 ]. Viral capsid stability can also impact innate immune sensing as altering favourable host-capsid interactions leads to premature uncoating [ 50 ] and exposure of viral intermediates in the cytosol where they can be recognized and trigger type I IFN in MDM [ 51 ] (Fig. 1 ).…”