Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins

Abstract

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.

How Viruses Use the VCP/p97 ATPase Molecular Machine

“…Experiments with WNV virus-like particles (VLPs) encoding a reporter gene revealed inhibition of early steps of infection with small interfering RNAs (siRNAs) specific for VCP [ 42 ]. These same siRNAs did not affect replication of VSV [ 42 ], a negative-stranded RNA virus, which also uses receptor-mediated endocytosis for cell entry [ 48 ]. Using a replication-defective reporter virus system to study entry and pre-replication events of multiple flaviviruses, an inhibitor of the ubiquitin-activating enzyme E1 also blocked YFV, ZIKV, and WNV uncoating [ 43 ].…”

Identification of Niemann-Pick C1 protein as a potential novel SARS-CoV-2 intracellular target

“…This highlights the specificity of the interaction and the inhibitor drugs thereby. In addition to this, another assay testing the compounds was performed using VSV, which needs an early/less acidic endosomes as reported ( Lay Mendoza et al, 2020 ; White and Whittaker, 2016 ). As expected, no severe impact was observed in the infection compared to HCoV-229E-GFP and SARS-CoV-2 ( Fig.…”

Modulation of immunosuppressant drug treatment to improve SARS-CoV-2 vaccine efficacy in mice

“…Individual mouse sera (n = 4) were heat inactivated (56 °C, 30 min) and serial diluted in Dulbecco’s Modified Eagle Medium (DMEM). Diluted sera were mixed with approximated 400 infectious particles of a recombinant vesicular stomatitis virus encoding the SARS-CoV-2 spike protein and nano-luciferase (room temperature, 30 min) [37] , [38] . To improve spike incorporation onto rVSV particles the cytoplasmic tail was removed.…”