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We designed a reverse genetics platform for production of SARS-CoV-2 based on a mammalian-expression BAC encoding the complete virus genome, including insertion of a codon-optimized nano-luciferase (nLuc) gene. This allows production of reporter virus by direct transfection into cells or by transfection of in vitro synthesized viral RNA.
To examine the role of Envelope (E) in SARS-CoV-2 infection, the E gene was deleted in the SARS-CoV-2 genome without disruption of expression of the other viral genes. For complementation of the deleted E gene in trans, we also engineered an inducible system to produce codon-optimized E.
dE SARS-CoV-2 is replication competent but attenuated compared to wild-type virus in multiple cell types in the absence of E expression. This was due to reduced virion production and those viruses appeared misshapen and lacked Spike (S) incorporation. While WT virus has been shown to produce syncytia, dE SARS-CoV-2 replication occurred exclusively by cell-to-cell transmission. Neutralizing antibodies were less effective in inhibiting transmission of dE SARS-CoV-2 compared to WT virus.
The mechanism of dE SARS-CoV-2 cell-to-cell transmission was due to greater S expression on the cell surface during infection rather than in the ER-Golgi compartment, suggesting E retains S at the site of virion production. In addition, without E expression, SARS-CoV-2 infection led to fragmentation of the ER-Golgi compartment, which was not observed in uninfected cells or cells infected with WT virus.
A better understanding of how E influences SARS-CoV-2 replication could guide directed design of novel therapeutics for treatment of COVID-19, as well as the potential for pan-coronavirus protection against future coronavirus outbreaks.
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