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

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 IC₅₀ 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 ³H-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.