Cystatin D, a natural salivary cysteine protease inhibitor, inhibits coronavirus replication at its physiologic concentration

Collins, A. R.; Grubb, A.

This study was conducted to examine the effect of cystatin D, a newly discovered salivary cysteine protease inhibitor, on human coronavirus replication. When MRC‐5, human diploid lung cells, were incubated with dilutions of recombinant human cystatin D from 0.65–10 μM for 1 h prior to, during and after infection with coronavirus OC43 and 229e strains, a decrease in virus yield was observed resulting in an IC(50) of 0.8 μM for both virus strains. This dose is within the normal concentration range of cystatin D, 0.12–1.9 μM found in saliva. When a single dose, 2.5 μM, was applied, cystatin inhibition of release of virus progeny was not overcome until three days post infection whereas inhibition by leupeptin, a serine and cysteine protease inhibitor, was completely abrogated by two days. When cellular toxicity was measured by (3)H‐thymidine uptake, cystatin D did not markedly affect cell proliferation below a 10 μM dose. The results demonstrate that cystatin D is a potent inhibitor of coronavirus replication.