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.
Highlights
Enveloped viruses are covered in a lipid membrane acquired by budding from infected cells.In order for enveloped viruses to infect a cell, the viral membrane must fuse with the cellular membrane, creating a pore through which the viral genome enters the cell cytoplasm
We produced a panel of recombinant vesicular stomatitis viruses
The production of replication competent chimeric vesicular stomatitis virus (VSV) particles has been used extensively to examine the virus entry of highly pathogenic viruses in a BLS2 system [62,63,64]. These particles are being used as vaccine vectors, with the recombinant VSV (rVSV)-ZEBOV becoming the first licensed EBOV vaccine [65,66]
Summary
Enveloped viruses are covered in a lipid membrane acquired by budding from infected cells. In order for enveloped viruses to infect a cell, the viral membrane must fuse with the cellular membrane, creating a pore through which the viral genome enters the cell cytoplasm. Viruses produce fusion proteins studded in their membrane. There are three defined classes of viral fusion proteins, termed class I, II and III [1,2]. While all proteins from all three classes are capable of forming fusion pores between the viral and cellular membranes, they have different properties and requirements [1,2,3]. We produced a panel of recombinant vesicular stomatitis viruses (rVSV)
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