Entry, replication and innate immunity evasion of BANAL-236, a SARS-CoV-2-related bat virus, in Rhinolophus and human cells

Asian Rhinolophus bats are considered the natural reservoirs of an ancestral SARS-CoV-2. However, the biology of SARS-CoV-2-related viruses in bat cells is not well understood. Here, we investigated the replication of BANAL-236, the only bat-derived SARS-CoV-2 relative isolated to date, in Rhinolophus cells. BANAL-236 did not replicate in wild-type Rhinolophus cell lines. Entry assays using pseudoviruses expressing the spike proteins (S) of SARS-CoV-2, BANAL-236, and BANAL-52 revealed that efficient S-mediated entry depends on the expression of human ACE2 (hACE2) and human TMPRSS2 (hTMPRSS2) in human and Rhinolophus cells. Expression of Rhinolophus entry factors, either alone or in combination, did not facilitate SARS-CoV-2 or BANAL-236 entry in human cells, suggesting that the S protein of BANAL-236 interacts more efficiently with hACE2 than with its Rhinolophus counterpart (rACE2). Through biochemical, virological, and electron microscopy analyses, we showed that BANAL-236 and SARS-CoV-2 completed their replication cycles in a Rhinolophus cell line engineered to express high levels of hACE2 and hTMPRSS2. Despite efficient viral replication in modified Rhinolophus and human cells, no induction of interferon (IFN)-stimulated genes was detected. Using a screening approach, we identified several BANAL-236 proteins that antagonize IFN production and signaling in human cells. Our findings thus show that BANAL-236 possesses critical features that enabled zoonotic spillover: hACE2 usage and potent evasion of human IFN responses. The Rhinolophus cellular model we established offers a platform for further investigating the interactions between bat coronaviruses and their reservoir hosts.