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Vesicular stomatitis virus (VSV) is an enveloped negative-stranded RNA virus that infects a wide range of animals and less frequently humans causing mild flu-like symptoms ( 15– 17). Therefore, pseudotyping viral systems have been widely employed to study highly infectious and pathogenic viruses such as Ebola virus, Middle Eastern Respiratory Syndrome (MERS) virus, or SARS viruses ( 12– 14). The use of replication-restricted pseudoviruses bearing viral coat proteins represents a safe and useful method that has been widely adopted by virologists to study viral entry, detection of nAbs in serum samples, and therapeutic development under less stringent biosafety conditions. On the other hand, experiments with actual pathogenic SARS-CoV-2 demands strict biosafety level-3 (BSL-3) laboratory conditions ( 11). However, the Spike protein alone fails to mimic the virus-host cell interaction.
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In vitro cellular assays using the Spike glycoprotein are therefore used to study viral infection or Spike/hACE2 interaction. Recovered COVID-19 patients develop neutralizing antibodies (nAbs) against the Spike glycoprotein proving its high antigenicity ( 9, 10). This S protein cleavage triggers conformational rearrangements of the S2 domain to activate the protein for viral membrane fusion and release of the viral genetic material into the cells ( 8, 9). Spike protein cleavage between the S1 and S2 domain is mediated by several host proteases, such as Furin or the transmembrane protease serine protease-2 (TMPRSS2). Once bound to hACE2, the S1 domain is cleaved and dissociated from the protein exposing the S2 domain containing the S transmembrane domain, cytoplasmic tail, and the fusion peptide ( 8). The S protein interacts with hACE2 through the receptor binding domain (RBD) located in the NTD (S1) ( 7). Coronavirus S glycoprotein is a class I viral fusion protein ( 5) containing a homotrimeric conformation with two functional subunits termed S1 and S2 present in the N-terminal domain (NTD) and C-terminal domain (CTD), respectively ( 6). The spike (S) glycoprotein is present on the surface of SARS-CoV-2 and is responsible for the viral tropism through host cell recognition by binding to the human angiotensin-converting enzyme 2 (hACE2) receptor ( 3, 4).
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Understanding the viral entry process is key for the development of therapeutic strategies to prevent viral spread. SARS-CoV-2 is an enveloped, positive-sense single stranded RNA virus belonging to the Betacoronavirus genus of the Coronaviridae family of viruses ( 2). The coronavirus disease (COVID-2019) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ( 1). This system is efficient (virus generation, titration, and infection assays can be performed in 1 week), quantitative, inexpensive, and readily scalable for application in drug development and therapeutic screening approaches. In addition, we have used this system to show infection of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We show that the system works efficiently in three unrelated, clinically relevant cell lines: human 293T (renal epithelial) cells, human Calu-3 (lung epithelial) cells, and the non-human primate (African Green Monkey) cell line, Vero-E6 (renal epithelial) cells. We present results of our optimization of the system to enhance viral infection levels through the over-expression of human ACE2 receptor and the overexpression of at least one of two proteases - TMPRSS2 or Furin, as well as, supplementation with Poloxamer 407 (P407) and Prostaglandin E2 (PGE2) as adjuvants. We describe the infection assay which uses GFP fluorescence as a measure of infection in a 24-well live imaging system.
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By incorporating the most up-to-date knowledge, we have developed a detailed, easy-to-follow novel protocol for producing SARS-CoV-2 spike-bearing pseudovirus using the VSV-ΔG system. The production of pseudotyped viral particles may seem like a daunting task for a non-virology laboratory without experience in the two most commonly used pseudotyping systems, namely retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVΔG). This is particularly important for patients who are at high risk for severe outcomes related to COVID-19. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood.