【病毒外文文獻】1998 Cystatin D, a natural salivary cysteine protease inhibitor, inhibits coronavirus replication at its physiologic con

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Oral Microbiol Immunoll998 13 59 61 Printed in Denmark AN rights reserved Copyright 6 Munksgaard 1998 mMKlalnral qy d ISSN 0902 0055 Short communication Cystatin D a natural salivary cysteine protease inhibitor inhibits coronavirus replication at its physiologic concentration A R Collins A Grubb2 State University of New York at Buffalo USA 2University of Lund Sweden Collins AR Grubb A Cystatin D a natural salivary cysteine protease inhibitor inhibits coronavirus replication at its physiologic concentration Oral Microbiol Immunoll998 13 5941 0 Munksgaard 1998 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 pM for I h prior to during and after infection with coronavirus OC43 and 229e strains a decrease in virus yield was observed resulting in an ICs0 of 0 8 pM for both virus strains This dose is within the normal concentration range of cystatin D 0 12 1 9 pM found in saliva When a single dose 2 5 pM 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 ceh lar toxicity was measured by 3H thymidine uptake cystatin D did not markedly affect cell proliferation below a 10 pM dose The results demonstrate that cysta tin D is a potent inhibitor of coronavirus replication Key words cystatins human coronavirus inhibition Arlene R Collins Department of Microbiology State University of New York at NY 14214 USA Accepted for publication June 3 1997 Cystatin D is the most recently de scribed human cystatin that in contrast to other family I1 cystatins has a very restricted tissue distribution comprising only salivary and lachrymal glands 1 3 6 The normal concentration range of cystatin D in mixed saliva is 0 12 1 9 pM 6 It has been suggested that cystatin D in saliva might play a protec tive role against potentially harmful ef fects of proteinases of bacterial fungal viral and cellular origin and thus could be considered a component of the non immune protective system in this cavity Several investigators have obtained evi dence for an antiviral effect on her pesvirus and coronavirus by cystatin C the most widely distributed family I1 cystatin with particularly high concen trations in seminal plasma and cerebro spinal fluid 1 4 5 Also inhibition of poliovirus by chicken cystatin has been reported 10 Both coronavirus and poliovirus express viral cysteine pro teinases that play important roles in replication by processing the polyprote ins translated from the large open read ing frames of these viruses 8 10 Therefore a direct effect of cystatin on the viral proteinases has been suggested lo although the mechanism of cellu lar uptake of the inhibitor is unclear 4 We report here that recombinant hu man cystatin D inhibits human cor onavirus replication as was previously shown for cystatin C and also that cys tatin D is more effective than leupeptin in slowing the release of virus from in fected cells Recombinant cystatin D was pre pared using Escherichia coli expression vector p cystatin D Arg containing a temperature sensitive repressor gene the phage PR promoter an optimized ribosome binding site the E coli outer membrane protein A signal peptide en coding sequence followed in frame by an Ar 6 cystatin D encoded cDNA devoid of its signal sequence and the phage fd transcription terminator 3 6 Isolation of Arg26 cystatin D from periplasmic extracts was accomplished by immunosorption and gel filtration 3 Lyophilized salt free Arg26 cystatin D was dissolved directly in Eagle s mini mum essential medium and used at con centrations from 10 0 65 pM to treat duplicate monolayer cultures of MRC 5 cells Viro Med Minnetonka MN about 140 000 cells per well for 1 h be fore during 1 h virus adsorption and during maintenance after infection in a yield reduction assay as described 5 Stock human coronavirus strains OC43 and 229e prepared from super natant medium of infected MRC 5 60 Collins a brief review Rev Med Virol 1994 4 35 46 Sadoul R Fernandez PA Quiquerez AL et al Involvement of the proteasome in the programmed cell death of NGF de prived sympathetic neurons EMBO J Taugner R Buhrle CP Nobiling R Kirschke H Coexistence of renin and ca thepsin B in epitheloid cell secretory granules Histochemistry 1995 83 103 108 Schroder E Phillips C Garman E Harlos K Crawford C X ray crystallo graphic structure of a papain leupeptin complex FEBS Lett 1993 315 38 42 1996 15 3845 3852