Development of a virus particle fusion assay to study and target virus entry

Virus entry leads to the delivery of the viral genome into the target cell for replication and viral gene expression. This critical step is therefore the target of many host immune mechanisms, as well as antiviral drugs. Various experimental systems have been developed to investigate virus entry and test virus entry inhibitors. Often, these systems are either cell-based or employ recombinant proteins to characterize the interaction between viral glycoproteins and their receptors, as well as determine the structural implications of virus attachment and entry. In this study, we have developed a quantitative virus particle fusion assay that allows for the measurement of viral membrane fusion events triggered by the binding of viral glycoproteins to their receptors. Specifically, we produced HIV-1 lentiviral particles incorporating HIV-1-Env/LgBiT-Vpr or CD4-CCR5/HiBiT-Vpr and recorded high levels of reconstituted NanoLuc luminescence when these virus particles are co-incubated. Background levels of luminescence were detected when these virus particles did not carry Env or CD4-CCR5, indicating that HIV-1 Env and CD4/CCR5 are essential for particle fusion and NanoLuc reconstitution. We further showed that HIV-1 entry inhibitors targeting Env, CD4, and CCR5, all potently inhibit lentiviral particle fusion. Lastly, the antiviral proteins SERINC5 and IFITM3, which are known to inhibit HIV-1 entry, also significantly diminish fusion in the context of this assay. Taken together, these data demonstrate that this quantitative lentiviral particle fusion assay can be used for discovering viral entry inhibitors, generating insights into the molecular mechanisms of virus entry, and elucidating the role of host factors in viral entry.