Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare glycoprotein-mediated entry efficiencies of: VSV G, SARS-CoV-2 S, EBOV GP, LASV GP, and CHIKV E we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase-PEST (NLucP) reporter gene, which we used in combination with the live-cell substrate Endurazine™ to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.