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Naihong Yan, Frank Piraino, Xuyang Liu; A Novel Antiviral Protein RC28. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6260.
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Herpes simplex keratitis is a major cause of adult eye disease, and may lead to chronic inflammation of the cornea. An antiviral protein, RC28, with anti-herpes virus activity was extracted from Rozites caperata (Cortinarious caperata). The goal of this study was to test the activity of RC28 in a murine herpes simplex virus (HSV)-1 keratitis model.
RC28 was isolated and purified from R. caperata mushroom. RC28 protein was sequenced from the amino terminus using the Edman degradation procedure. RC28 cDNA fragment was obtained using 3’- RACE method and expressed in E.coli and purified by affinity chromatography. The in vitro antiviral activity of RC-2 against HSV-1 was determined by yield reduction and viral inactivation assays. The virus keratitis in mice animal models had established to study the role of anti-virus RC28.
RC28 with anti-herpesvirus activity was purified from a PBS extract of Rozites caperata by acetone precipitation followed by gel filtration and ion exchange chromatography. The molecular weight of this protein was 28.251kDa as measured by MALDI-TOF mass spectrography. RC28 inhibited HSV-1 replication in vitro with an IC(50) value of 0.078mg/ml and a therapeutic index >32. The first 30 amino acid residues of RC28 were sequenced by Edman degradation to be MLTYR GKLNW YNYAV NEGFT LILPG XELKV. Based on that sequence, the RC28 cDNA fragment was cloned by 3'-RACE, and the rest of the amino acid sequence was inferred from the cDNA sequence. RC28 was cloned into pET26b(+) vector and expressed of protein in Escherichia Coli. Cytotoxicity test showed that the RC28 is very weak cytotoxicity and RC28 can inhibit HSV-1 in culture. The animal model results showed that RC28 delayed the occurrence of stromal keratitis and alleviated the severity of the disease.
RC28 is an antiviral protein with multi-functional activities that interfere with both early and late HSV-1 viral functions, a remarkable and clinically important characteristic of an antiviral drug.
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