Sillucin

Chilo iridescent virus (CIV), the type species of the genus Iridovirus, a member of the Iridoviridae family, is highly pathogenic for a variety of insect larvae. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity and strategy of the CIV genome was elucidated by the analysis of the complete DNA nucleotide sequence of the viral genome (212,482 bp) using cycle sequencing by primer walking technology. Both DNA strands were sequenced independently and the average redundancy for each nucleotide was found to be 1.85. The base composition of the viral genomic DNA sequence was found to be 71.37% A+T and 28.63% G+C. The CIV genome contains 468 open reading frames (ORFs). The size of the individual viral gene products ranges between 40 and 2432 amino acids. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs were nonoverlapping. The comparison of the deduced amino acid sequences to entries in protein data banks led to the identification of several genes with significant homologies, such as the two major subunits of the DNA-dependent RNA polymerase, DNA polymerase, protein kinase, thymidine and thymidylate kinase, thymidylate synthase, ribonucleoside-diphosphate reductase, major capsid protein, and others. The highest homologies were detected between putative viral gene products of CIV and lymphocystis disease virus of fish (LCDV). Although many CIV putative gene products showed significant homologies to the corresponding viral proteins of LCDV, no colinearity was detected when the coding strategies of the CIV and LCDV-1 were compared to each other. An intriguing result was the detection of a viral peptide of 53 amino acid residues (ORF 160L) showing high homology (identity/similarity: 60.0%/30.0%) to sillucin, an antibiotic peptide encoded by Rhizomucor pusillus. Iridovirus homologs of cellular genes possess particular implications for the molecular evolution of large DNA viruses.

The disulfide structure of sillucin, a highly knotted, cysteine-rich, antimicrobial peptide, isolated from Rhizomucor pusillus, has been determined to be Cys2--Cys7, Cys12--Cys24, Cys13--Cys30, and Cys14--Cys21 by disulfide mass mapping based on partial reduction and CN-induced cleavage enabled by cyanylation. The denatured 30-residue peptide was subjected to partial reduction by tris(2-carboxyethyl)phosphine hydrochloride at pH 3 to produce a mixture of partially reduced sillucin species; the nascent sulfhydryl groups were immediately cyanylated by 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate. The cyanylated species, separated and collected during reversed phase high-performance liquid chromatography, were treated with aqueous ammonia, which cleaved the peptide chain on the N-terminal side of cyanylated cysteine residues. The CN-induced cleavage mixture was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry before and after complete reduction of residual disulfide bonds in partially reduced and cyanylated species to mass map the truncated peptides to the sequence. Because the masses of the CN-induced cleavage fragments of both singly and doubly reduced and cyanylated sillucin are related to the linkages of the disulfide bonds in the original molecule, the presence of certain truncated peptide(s) can be used to positively identify the linkage of a specific disulfide bond or exclude the presence of other possible linkages.