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. 2016 Dec 5;6(23):e2035.
doi: 10.21769/BioProtoc.2035.

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Jean Kaoru Millet  1 , Gary R Whittaker  1
Affiliations

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Jean Kaoru Millet et al. Bio Protoc. .

Abstract

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay.

Keywords: Coronavirus; Envelope glycoprotein; Murine leukemia virus; Pseudotyped particle; Spike.

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Figures

Figure 1
Figure 1. HEK-293T/17 cell confluency before transfection
For efficient transfection and pseudotyped particle production, HEK-293T/17 cell confluency should be around the 40-60% range as shown in the above microscopy picture taken with a Zeiss Axiovert 200 microscope. Scale bar represents 100 μm.
Figure 2
Figure 2. Huh-7 cell confluency before infection with pseudotyped particles
For efficient transduction by pseudotyped particles, Huh-7 cells should be almost completely confluent as shown in the above microscopy picture taken with a Zeiss Axiovert 200 microscope. Scale bar represents 100 μm.
Figure 3
Figure 3. MERS-CoV S-pseudotyped particle infectivity assay in human liver cells (Huh-7)
Displayed are luciferase activity measurements of lysed Huh-7 cells 72 h post-infection for the following conditions: non-infected (n.i.), ‘bald’ (no envelope) pseudotyped particles (Δenvpp), VSV-G-pseudotyped particles (VSV-Gpp), and MERS-CoV S-pseudotyped particles (MERS-Spp). Experiments performed in triplicates and data presented is average relative luciferase units (log10) of three independent experiments (n = 3). Error bars indicate standard deviation (s.d.).
See this image and copyright information in PMC

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