The robust inflammatory responses caused by MjHKU4r-S mediated cell fusion

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has extremely high virulence and pathogenicity in human, but some MERS-related coronaviruses (bat-CoV HKU4, bat-CoV HKU5-1/2 and MOW15-22/PnNL2018B) showed limited infectivity in human beings. Unlike previously reported MERS-related coronaviruses, the currently identified MERS-related Manis javanica HKU4-related coronavirus (MjHKU4r-CoV-1) exhibited significant infectivity in human cell, organs, and hDPP4-transgenic mice. However, the detailed pathogenic features and underlying mechanisms were unknown. In virological experiments, we found MjHKU4r-CoV-1 triggered extensive syncytia formation by its spike protein (S) and caused significant inflammation responses. Herein, we use RNA sequencing (RNA-Seq) to investigate the transcriptional alterations underlying the robust inflammatory responses caused by S protein mediated syncytia formation. 293T cells were transfected with the pAAV-IRES-EGFP vector plasmid encoding S proteins of MjHKU4r-CoV-1, HKU4 (the negative control), or SARS-CoV-2 KP.2 variant (the positive control) S proteins to serve as effector cells. Caco-2 cells, which naturally express human ACE2 receptors on their membrane surface, were used as target cells. The effector cells were cultured, collected, resuspended, and subsequently co-incubated with the target cells (Caco-2) for 36 hours at 37 °C. Hence, three groups were set in our study: 293T-MjHKU4r-S:Caco2 (the study group); 293T-SARS-CoV-2-S:Caco2 (the positive control); 293T-HKU4-S:Caco2 (the negative control), and each group had four biological replications. Following incubation, cells were lysed, and RNA extraction was performed according to the manufacturer’s instructions. Purified RNA was used for cDNA synthesis and library preparation. RNA sequencing was conducted on the Illumina NovaSeq 6000 PE150 platform.