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Principal Investigator: Alex Compton
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2020
Award: $265,660
Funding agency: National Cancer Institute
We have successfully produced HIV-based pseudovirus bearing the spike protein of SARS-CoV-1 and SARS-CoV-2 and produced cell lines that are permissive to these pseudoviruses. We have developed a protocol for transiently transfecting human ACE2 (the receptor for SARS-CoV-1 and SARS-CoV-2) and TMPRSS2 (a protease that activates the fusion potential of SARS-CoV-1 and SARS-CoV-2 spike proteins) into HEK293T cells stably expressing human IFITM1, IFITM2, IFITM3, and mutants thereof. We have challenged these cells with the HIV-SARS pseudoviruses and found that the human IFITM proteins inhibit both SARS-CoV-1- and SARS-CoV-2-mediated entry into cells, albeit to different extents. Whereas IFITM3 strongly inhibits SARS-CoV-1-mediated entry, it only slightly inhibits that driven by SARS-CoV-2. Furthermore, if target cells express TMPRSS2, the inhibitory effect of IFITM3 is negligible. These results suggest that viruses utilizing TMPRSS2 have decreased sensitivity to IFITM proteins, indicating that TMPRSS2 usage may alter the virus entry route into the cell. Given this variable effect of ectopic IFITM protein expression, we assessed how IFITM proteins endogenously expressed in two cell lines that are naturally permissive to coronavirus infection (Caco-2 and Calu-3) affect pseudovirus infection. We found that siRNA-mediated knockdown of IFITM2 and IFITM3, but not IFITM1, led to enhanced infection by HIV-SARS-2 (about 3-fold). To complement our studies, we are collaborating with Jacob Yount at Ohio State University, who is challenging our cell lines with wild-type SARS-CoV-2.
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