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 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.

Chikungunya virus entry and infectivity is primarily facilitated through cell line dependent attachment factors in mammalian and mosquito cells

“…The amount of infectious virus was determined by calculating the 50% tissue culture infective dose (TCID 50 ) units per mL through end-point dilution using the Spearman-Karber method (Ramakrishnan, 2016). Recombinant vesicular stomatitis viruses containing the CHIKV, Ebola, or Lassa glycoproteins were generated as previously described (Lay Mendoza et al, 2020). Coxsackie B5 virus was amplified in VeroS cells.…”

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].…”

Untargeted Lipidomics of Vesicular Stomatitis Virus-Infected Cells and Viral Particles

“…Vero (Vervet monkey kidney) cells stably expressing SLAM were maintained at 37 • C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS. Recombinant VSV encoding the GFP or nano luciferase fused to a destabilization domain PEST (nLuciP) reporter gene was recovered from transfecting cells with the four plasmid VSV rescue system (a gift from Michael Whitt, University of Tennessee, Kerafast, 7753828) as previously described [24].…”