ORTHOPOXVIRUS FUSION INHIBITOR GLYCOPROTEIN SPI-3 (OPEN READING FRAMEK2L) CONTAINS MOTIFS CHARACTERISTIC OF SERINE PROTEINASE-INHIBITORS THAT ARE NOT REQUIRED FOR CONTROL OF CELL-FUSION

The cowpox virus (CPV) SPI-3 gene (open reading frame K2L in vaccinia virus) is one of three orthopoxvirus genes whose products are members of the serpin (serine proteinase inhibitor) superfamily, The CPV SPI-3 gene, when overexpressed by using the vaccinia virus/T7 expression sy stem, synthesized two proteins of 50 and 48 kDa. Treatment with the N glycosylation inhibitor tunicamycin converted the two SPI-3 proteins t o a single 40-kDa protein, close to the size of 42 kDa predicted from the DNA sequence, suggesting that the SPI-3 protein, unlike the other two orthopoxvirus serpins, is a glycoprotein. Immunoblotting with an a nti-SPI-3 antibody showed that the SPI-3 protein is synthesized early in infection prior to DNA replication. SPI-3 inhibits cell-cell fusion during infections with both CPV and vaccinia virus. A transfection as say was devised to test engineered mutants of SPI-3 for the ability to inhibit fusion. Two mutants,vith C-terminal deletions of 156 and 70 a mino acids were completely inactive in fusion inhibition. Site-directe d mutations were constructed near the C terminus of SPI-3, in or near the predicted reactive-site loop which is conserved in inhibitory serp ins. Substitutions within the loop at the P1 to P1' positions and P5 t o P5' positions, inclusive, did not result in any loss of activity, no r did changes at the P17 to P10 residues in the stalk of the reactive loop. Therefore, SPI-3 does not appear to control cell fusion by actin g as a serine proteinase inhibitor.