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Inhibition of the human complement component C4 by Flavivirus non-structural protein NS1
Abstracts / Molecular Immunology 47 (2010) 2198–2294
the E283A/E304A mutant having the greatest reduction in binding affinity. All modified fHbps were immunogenic and elicited high titres of antibodies demonstrated by ELISA. Additionally, sera raised against the modified fHbps demonstrated bactericidal activity equivalent to sera raised by wild type fHbp. These results indicate that modification of fHbp to impair fH binding does not affect the immunogenicity of this antigen, and modified fHbps are thus potential vaccine candidates. doi:10.1016/j.molimm.2010.05.126 203 Inhibition of the human complement component C4 by Flavivirus non-structural protein NS1 Panisadee Avirutnan a,d , Anja Fuchs a , Richard E. Hauhart a , Pawit Somnuke a,e , Soonjeon Youn a , Michael S. Diamond a,b,c , John P. Atkinson a,b,c a
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States b Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, United States c Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, United States d Medical Molecular Biology Unit, Department of Research and Development, Mahidol University, Bangkok-noi, Bangkok 10700, Thailand e Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok 10700, Thailand The complement system is a key player in innate and adaptive immune defense against many microorganisms. Viruses have evolved multiple strategies to avoid recognition by this cascade whose fragments opsonize, lyse and neutralize viruses. Flavivirus NS1 is a secreted non-structural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that Dengue (DENV), West Nile (WNV), and yellow fever (YFV) virus NS1 attenuate classical and lectin pathway via a direct interaction with C4. In three types of hemolytic assays employing sheep red blood cells (SRBCs) and purified human components or C4 deficient guinea pig serum, binding of NS1 to C4 reduced C4b deposition and therefore classical pathway C3 and C5 convertase activity and hemolysis. Although purified, recombinant NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient cleavage of C4 to C4b. Through this mechanism, NS1 was shown to protect DENV from complement-dependent neutralization in solution – the antagonistic effect of NS1 was C4-dependent and thus attenuated the classical and lectin pathways. This activity may limit the supply of C4 to participate in complement activation at the infectious site. Overall, these studies define a novel immune evasion mechanism for restricting complement control of a microbial infection. doi:10.1016/j.molimm.2010.05.128
2239
204 Complement evasion by vesicular stomatitis virus involves recruitment of host complement regulatory proteins John B. Johnson a , Douglas S. Lyles b , Griffith D. Parks a a
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, United States b Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, United States The complement system is a powerful arm of innate immunity during viral infections. The goal of the work described here was to determine if vesicular stomatitis virus (VSV) possesses mechanisms to limit complement-mediated neutralization. VSV is a prototypic negative strand RNA virus of the family Rhabdoviridae that is related to rabies virus and the newly emerging human pathogen Chandipura virus. Previously, VSV was shown to activate the complement classical pathway (CP) by an antibody-dependent mechanism which results in virus neutralization. Western blot (WB) analysis of VSV grown in human epithelial cell lines showed that membrane cofactor protein (CD46) and decay acceleration factor (CD55) were associated with purified particles. Immunogold electron microscopic (EM) analysis showed intense labeling for CD46 and CD55 around the virus envelope, suggesting that these cofactors were incorporated into the viral envelope. To test the functional consequences of cofactor incorporation, cofactor activity assays (CFA) were reconstituted in vitro with purified C3b/C4b, factor I and VSV. Cofactor activity supplied by the VSV particles was capable of mediating the complete and rapid inactivation of C3b into iC3b. Interestingly, C4b was also completely converted to C4c and C4d. Since CD46 is a weak cofactor for C4b cleavage and VSV activates the CP, we hypothesized that VSV could recruit a CP-specific regulatory protein that has much greater activity for C4b. WB analysis and immunogold labeling of purified virus suggested that C4b binding protein (C4BP) was associated with VSV particles. Our findings support the hypothesis that VSV recruits both cell-associated and soluble complement regulatory proteins to limit neutralization. These complement regulatory proteins may thus contribute to viral pathogenesis. doi:10.1016/j.molimm.2010.05.129 205 Intact complement C3 binds to Staphylococcus aureus in hyperglycemic conditions without complement activation Pamela Hair, Kenji Cunnion Eastern Virginia Medical School, United States
Background and rationale: Diabetes affects 23.6 million people in the U.S., of whom approximately 20% will suffer wound infections. In 2004, diabetics underwent 71,000 non-traumatic limb amputations. In diabetics, Staphylococcus aureus is the most common cause of foot infections and a major pathogen in severe polymicrobial lower extremity infections. The complement system plays a vital role in innate humoral immunity and its role in controlling S. aureus infections in hyperglycemic environment remains poorly understood. Methods: The binding of C3, the central component of the complement system, to S. aureus isolates was measured in euglycemic and hyperglycemic environments for human serum and purified C3. Results: C3 binding to S. aureus increased 5-fold in hyperglycemic conditions (17 mM glucose) compared with euglycemia
the E283A/E304A mutant having the greatest reduction in binding affinity. All modified fHbps were immunogenic and elicited high titres of antibodies demonstrated by ELISA. Additionally, sera raised against the modified fHbps demonstrated bactericidal activity equivalent to sera raised by wild type fHbp. These results indicate that modification of fHbp to impair fH binding does not affect the immunogenicity of this antigen, and modified fHbps are thus potential vaccine candidates. doi:10.1016/j.molimm.2010.05.126 203 Inhibition of the human complement component C4 by Flavivirus non-structural protein NS1 Panisadee Avirutnan a,d , Anja Fuchs a , Richard E. Hauhart a , Pawit Somnuke a,e , Soonjeon Youn a , Michael S. Diamond a,b,c , John P. Atkinson a,b,c a
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States b Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, United States c Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, United States d Medical Molecular Biology Unit, Department of Research and Development, Mahidol University, Bangkok-noi, Bangkok 10700, Thailand e Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok 10700, Thailand The complement system is a key player in innate and adaptive immune defense against many microorganisms. Viruses have evolved multiple strategies to avoid recognition by this cascade whose fragments opsonize, lyse and neutralize viruses. Flavivirus NS1 is a secreted non-structural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that Dengue (DENV), West Nile (WNV), and yellow fever (YFV) virus NS1 attenuate classical and lectin pathway via a direct interaction with C4. In three types of hemolytic assays employing sheep red blood cells (SRBCs) and purified human components or C4 deficient guinea pig serum, binding of NS1 to C4 reduced C4b deposition and therefore classical pathway C3 and C5 convertase activity and hemolysis. Although purified, recombinant NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient cleavage of C4 to C4b. Through this mechanism, NS1 was shown to protect DENV from complement-dependent neutralization in solution – the antagonistic effect of NS1 was C4-dependent and thus attenuated the classical and lectin pathways. This activity may limit the supply of C4 to participate in complement activation at the infectious site. Overall, these studies define a novel immune evasion mechanism for restricting complement control of a microbial infection. doi:10.1016/j.molimm.2010.05.128
2239
204 Complement evasion by vesicular stomatitis virus involves recruitment of host complement regulatory proteins John B. Johnson a , Douglas S. Lyles b , Griffith D. Parks a a
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, United States b Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, United States The complement system is a powerful arm of innate immunity during viral infections. The goal of the work described here was to determine if vesicular stomatitis virus (VSV) possesses mechanisms to limit complement-mediated neutralization. VSV is a prototypic negative strand RNA virus of the family Rhabdoviridae that is related to rabies virus and the newly emerging human pathogen Chandipura virus. Previously, VSV was shown to activate the complement classical pathway (CP) by an antibody-dependent mechanism which results in virus neutralization. Western blot (WB) analysis of VSV grown in human epithelial cell lines showed that membrane cofactor protein (CD46) and decay acceleration factor (CD55) were associated with purified particles. Immunogold electron microscopic (EM) analysis showed intense labeling for CD46 and CD55 around the virus envelope, suggesting that these cofactors were incorporated into the viral envelope. To test the functional consequences of cofactor incorporation, cofactor activity assays (CFA) were reconstituted in vitro with purified C3b/C4b, factor I and VSV. Cofactor activity supplied by the VSV particles was capable of mediating the complete and rapid inactivation of C3b into iC3b. Interestingly, C4b was also completely converted to C4c and C4d. Since CD46 is a weak cofactor for C4b cleavage and VSV activates the CP, we hypothesized that VSV could recruit a CP-specific regulatory protein that has much greater activity for C4b. WB analysis and immunogold labeling of purified virus suggested that C4b binding protein (C4BP) was associated with VSV particles. Our findings support the hypothesis that VSV recruits both cell-associated and soluble complement regulatory proteins to limit neutralization. These complement regulatory proteins may thus contribute to viral pathogenesis. doi:10.1016/j.molimm.2010.05.129 205 Intact complement C3 binds to Staphylococcus aureus in hyperglycemic conditions without complement activation Pamela Hair, Kenji Cunnion Eastern Virginia Medical School, United States
Background and rationale: Diabetes affects 23.6 million people in the U.S., of whom approximately 20% will suffer wound infections. In 2004, diabetics underwent 71,000 non-traumatic limb amputations. In diabetics, Staphylococcus aureus is the most common cause of foot infections and a major pathogen in severe polymicrobial lower extremity infections. The complement system plays a vital role in innate humoral immunity and its role in controlling S. aureus infections in hyperglycemic environment remains poorly understood. Methods: The binding of C3, the central component of the complement system, to S. aureus isolates was measured in euglycemic and hyperglycemic environments for human serum and purified C3. Results: C3 binding to S. aureus increased 5-fold in hyperglycemic conditions (17 mM glucose) compared with euglycemia