(197j) Investigating the Role of Calcium on Coronavirus Fusion with Host Cells

Coronaviruses (CoVs) are a diverse family of enveloped viruses responsible for causing respiratory and/or enteric diseases across a wide range of species, causing widespread outbreaks in the human population. The expansive animal host range of CoVs can be attributed to their ability to adapt to diverse cellular environments (i.e. pH, ions, proteases, receptors), and exploit different entry pathways to mediate viral-host cell membrane fusion and infect cells. Ions have arisen as an important factor in the viral membrane fusion mechanism. Within the CoV family, our team has found that calcium availability leads to increased virus infection of cells. Previously, we observed that calcium availability also promotes insertion of SARS FP into the host cell bilayer and subsequent membrane lipid ordering, pointing to the possible role of calcium in interacting directly with the FP during CoV membrane fusion activity.

Due to the highly conserved nature of the FP between SARS- and MERS-CoV, we chose to investigate the specific binding pockets of Ca²⁺ within SARS- and MERS-CoV. We used site-directed mutagenesis and infectivity assays to pinpoint specific residues that lead to changes in infectivity when calcium is present or not. We identified potential calcium-binding residues by substituting the charged residues (i.e. aspartic acid, glutamic acid) in the FP with non-charged amino residue, alanine. We compared the infectivity of mutant and wild-typed CoV VLPs under calcium-rich or poor environments using available FDA-approved calcium blocking drugs. We further show that this enhancement could be attributed to Ca²⁺ increasing SARS- and MERS-CoV FP fusion-relevant membrane ordering. Although both SARS-CoV and MERS-CoV FP interact with Ca²⁺, our data suggests differences in their interactions. SARS-CoV FP can bind two Ca²⁺, whereas MERS-CoV FP can only bind one Ca²⁺. Overall, these results demonstrate that Ca²⁺ have specific interactions with CoV FP.