Date Published: September 30, 2016
Publisher: Public Library of Science
Author(s): Claire Da Silva Santos, Kevin Tartour, Andrea Cimarelli, Michael Emerman.
To better characterize the behavior of HIV-1 capsids we developed EURT, for Entry/Uncoating assay based on core-packaged RNA availability and Translation. EURT is an alternative to Blam-Vpr, but as reporter RNA translation relies on core opening, it can be used to study viral capsids behavior. Our study reveals the existence of two major capsid species, a dead end one in which the viral genome is readily exposed to the cytoplasm and a functional one in which such exposure requires artificial core destabilization. Although reverse transcription drives a faster loss of susceptibility of viral cores to high doses of PF74, it does not lead to higher exposure of the viral genome, implying that viral cores protect the genome irrespectively of reverse transcription. Lastly, IFNα drifts cores from functional to non-functional species, revealing a novel core-destabilizing activity. This assay sheds new light on the behavior of viral cores inside target cells.
The correct entry and migration of viral genomes, as well as their protection from assaults driven by cellular effectors represent key steps for the successful infection of target cells. In the case of the HIV-1 lentivirus, the binding of the viral envelope protein gp120 to its cellular receptor CD4 and its co-receptors CCR5 or CXCR4 triggers the fusion between the viral and the cell membranes. This event leads to the cytoplasmic release of viral nucleoprotein complexes that contain the viral genome, as well as viral and cellular proteins (VNCs, more often referred to as cores, or capsids).
In the work presented here, we aimed in the first instance at developing a novel HIV-1 entry assay based on a viral genomic RNA mimic delivered and directly translated into the cell cytoplasm after viral to cellular membrane fusion. The data we present here indicate that in its easiest setting, this assay is highly sensitive, it is linear and yields virtually no background when HIV-1 viral particles are produced and purified as described here. Side-by-side comparison with the more widely used Blam-Vpr based test  points to the EURT assay as a valid alternative to study viral entry, at least for most applications (see further considerations below). We believe that in light of the fact that it uses a luciferase-based readout, the assay developed here may be easier to handle large screens that aim at identifying novel inhibitors of HIV-1 entry.