The cowpox virus serpin SPI-3 complexes with and inhibits urokinase-type and tissue-type plasminogen activators and plasmin.
The orthopoxvirus serpin SPI-3 is N-glycosylated and suppresses fusion between infected cells. Although SPI-3 contains motifs conserved in inhibitory serpins, no proteinase inhibition by SPI-3 has been demonstrated, and mutations within the serpin reactive center loop (RCL) do not affect the ability to regulate cell fusion. We demonstrate here that SPI-3 protein expressed by transcription/translation in vitro is able to form SDS-stable complexes with the serine proteinases plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), consistent with inhibitory activity of the serpin. Weaker complexes were noted with factor Xa and thrombin. Mutation of Arg-340/Ser-341 at the predicted P1/P1' sites within the RCL prevented the formation of complexes between SPI-3 and plasmin, uPA, or tPA, suggesting that the arginine at the P1 position was required for complex formation. SPI-3 protein lacking the N-terminal signal peptide was purified by means of an N-terminal His(10)-tag and gave complete inhibition in vitro of plasmin, uPA, and tPA and partial inhibition of factor Xa. SPI-3 is therefore a bifunctional protein that acts as a proteinase inhibitor and suppresses infected cell-cell fusion. As a proteinase inhibitor, SPI-3 has similar specificity to the leporipoxvirus SERP1 protein of myxoma virus, although the two serpins are less than 30% identical overall. The inhibition constants of SPI-3 for plasmin, uPA, and tPA were determined to be 0.64, 0.51, and 1.9 nM, respectively, very similar to the corresponding K(i) values of SERP1.
Turner
P C
PC
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, 32610-0266, USA.
Baquero
M T
MT
Yuan
S
S
Thoennes
S R
SR
Moyer
R W
RW
D000209:Acute-Phase Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D002460:Cell Line; D015608:Cowpox virus / Q000201:enzymology*; D005341:Fibrinolysin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D009233:Myxoma virus / Q000201:enzymology; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000502:physiology*; D015843:Serpins / Q000502:physiology*; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins / Q000502:physiology*; D000979:alpha-2-Antiplasmin / Q000378:metabolism
D000209:Acute-Phase Proteins; D046911:Macromolecular Substances; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D014764:Viral Proteins; D000979:alpha-2-Antiplasmin; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator