Background

Viral pseudotyped particles are very useful tools for studying the entry pathways that enveloped viruses use and for generating novel gene-delivery vectors. These synthetic enveloped viruses are derived from a parental virus, usually a rhabdovirus or a retrovirus, which forms the core of the particle that can incorporate in its membrane a wide range of viral envelope glycoproteins from heterologous viruses. Several model viruses such as the retroviral murine leukemia virus (MLV) and human immunodeficiency virus-1 (HIV-1) or the rhabdoviral vesicular stomatitis virus (VSV) have been successfully used to generate viral pseudotyped particles (also named pseudoviruses or pseudovirions). Virus pseudotyping is particularly useful for the following scenarios: (i) to quantify the viral entry process of enveloped viruses using reporter genes like green fluorescent protein (GFP) or luciferase, (ii) to study host cell entry of enveloped viruses that cannot be cultivated in cell culture, (iii) to study entry pathways of risk group (RG) 3 or 4 viral pathogens when biosafety level (BSL) 3 or 4 facilities are not available, (iv) to generate cells stably expressing a specific gene of interest or for specific gene silencing, (v) to produce vectors for gene delivery allowing control over cell tropism. Pseudotyped particles can be used to complement native virus infection assays, especially regarding study of virus entry events.

The protocol described here is highly adaptable both in terms of scale of production and type of envelope glycoprotein that can be incorporated. It has been extensively used in our research on viral entry of various enveloped viruses, including VSV (Sun et al., 2008), influenza virus (Tse et al., 2014) and coronaviruses (Belouzard et al., 2009; Millet and Whittaker 2014; Millet et al., 2016). We have successfully used this method to pseudotype viral envelope glycoproteins from all three classes of viral fusion proteins: influenza hemagglutinin (HA, class I), coronavirus spike (S, class I), Ebola glycoprotein (GP, class I), Semliki forest virus (SFV) E1 (class II), and vesicular stomatitis virus (VSV) G glycoprotein (class III). The technique described here is based on work performed by Bartosch and colleagues (Bartosch et al., 2003), and employs the so-called ‘three-plasmid’ co-transfection strategy in which producer HEK-293T/17 cells are co-transfected with the following plasmids: a plasmid allowing expression of MLV retroviral core genes gag and pol but lacking the MLV envelope glycoprotein-encoding env gene, a transfer vector containing a luciferase reporter gene flanked by retroviral regulatory LTR regions and a packaging signal, along with a plasmid allowing expression of the desired envelope glycoprotein. The co-expression of these three plasmids allows synthesis of LTR-flanked reporter gene-containing RNA, MLV-derived proteins and heterologous envelope glycoprotein. During pseudotyped particle formation, which occurs at the plasma membrane, the RNAs containing the LTR-flanked luciferase gene get incorporated into nascent particles formed by assembly and budding of MLV capsid proteins that also recruit heterologous viral envelope glycoproteins. Upon infection in susceptible cells, the pseudotyped virus entry pathway is solely governed by the heterologous virus envelope glycoprotein used. As such, pseudovirions are excellent surrogates to study the entry pathway of enveloped viruses. Once virus entry has occurred, the pseudotyped virus RNAs are released in the cell and the retroviral reverse transcriptase and integrase then reverse transcribe the molecules into double stranded DNA and integrate them into the genome of target cells. Because the sequence that gets integrated only contains the gene encoding the luciferase reporter but none of the MLV genes, the pseudotyped particles are inherently safer as they only allow for one round of infection. After infection, a simple luciferase assay allows quantification of infectivity of the pseudotyped particle studied.

The following protocol can form the basis of useful experiments for the study of the heterologous envelope glycoprotein function during virus entry, for example by performing the infection assay using different infection conditions such as receptor/co-receptor expression in target cells, virus binding time and temperature, pH, endocytosis inhibitors, protease inhibitors, neutralizing antibodies, etc.