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

Viruses. 2020 Dec 17;12(12):1457. doi: 10.3390/v12121457.


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.

Keywords: chikungunya; coronavirus; ebola; entry; kinetics; lassa; live assay; luciferase; real-time; vesicular stomatitis virus.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • Chikungunya virus / genetics
  • Chlorocebus aethiops
  • Cloning, Molecular
  • Ebolavirus / genetics
  • Gene Expression*
  • Gene Order
  • Genes, Reporter
  • Genetic Vectors / genetics*
  • Glycoproteins / genetics*
  • Humans
  • Lassa virus / genetics
  • SARS-CoV-2 / genetics
  • Time Factors
  • Vero Cells
  • Vesicular stomatitis Indiana virus / genetics*
  • Viral Envelope Proteins / genetics*
  • Virus Internalization*
  • Virus Replication


  • Glycoproteins
  • Viral Envelope Proteins