Human astrovirus coat protein binds C1q and MBL and inhibits the classical and lectin pathways of complement activation
Department of Pediatrics, Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, VA 23510, United States. Molecular Immunology
(Impact Factor: 2.97).
11/2009; 47(4):792-8. DOI: 10.1016/j.molimm.2009.10.006
Human astroviruses (HAstVs) constitute a family of non-enveloped, RNA viruses which cause infantile gastroenteritis. We have previously demonstrated that purified HAstV coat protein (CP), multiple copies of which compose the viral capsid, bind C1q resulting in inhibition of classical complement pathway activity. The objective of this study was to further analyze the mechanism by which CP inhibits C1 activation. CP inhibited C1 activation, preventing cleavage of C1s to its active form in the presence of heat-aggregated IgG, a potent classical pathway activator. CP also inhibited generation of the potent anaphylatoxin C5a. CP dose-dependently bound to C1q, the isolated globular heads and the collagen-like regions of the C1q molecule. When CP was added to C1, C1s dissociated from C1q suggesting that CP functionally displaces the protease tetramer (C1s-C1r-C1r-C1s). Given the structural and functional relatedness of C1q and MBL, we subsequently investigated the interactions between CP and MBL. CP bound to purified MBL and was able to inhibit mannan-mediated activation of the lectin pathway. Interestingly, CP did not bind to a variant of MBL that replaces a lysine residue (Lys55) critical for binding to MASP-2, a functional homolog of C1s. Finally, CP was shown to cross the species barrier to inhibit C3 activation and MAC formation in rat serum. These findings suggest CP inhibits C1 and MBL activation via a novel mechanism of interference with the normal interaction of the recognition molecule with its cognate serine proteases.
Available from: Michael P Stolz
- "A Western blot to assess C1s activation by heat aggregated IgG has been previously described [27,28] One microliter of partially purified C1 (0.2 mg/ml, Complement Technologies, Inc.), 5 μl of heat-aggregated IgG (1:250 dilution from 50 μg/ml stock), and 5 μl of PBS were combined on ice and samples incubated at the following temperatures: 31, 33, 35, 37, 39 and 41°C for 0, 30, 45, 60 and 90 minutes. After incubation, 4 μl of loading buffer was added and each sample was boiled. "
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Therapeutic hypothermia is a treatment modality that is increasingly used to improve clinical neurological outcomes for ischemia-reperfusion injury-mediated diseases. Antibody-initiated classical complement pathway activation has been shown to contribute to ischemia-reperfusion injury in multiple disease processes. However, how therapeutic hypothermia affects complement activation is unknown. Our goal was to measure the independent effect of temperature on complement activation, and more specifically, examine the relationship between clinical hypothermia temperatures (31–33°C), and complement activation.
Antibody-sensitized erythrocytes were used to assay complement activation at temperatures ranging from 0-41°C. Individual complement pathway components were assayed by ELISA, Western blot, and quantitative dot blot. Peptide Inhibitor of complement C1 (PIC1) was used to specifically inhibit activation of C1.
Antibody-initiated complement activation resulting in eukaryotic cell lysis was increased by 2-fold at 31°C compared with 37°C. Antibody-initiated complement activation in human serum increased as temperature decreased from 37°C until dramatically decreasing at 13°C. Quantitation of individual complement components showed significantly increased activation of C4, C3, and C5 at clinical hypothermia temperatures. In contrast, C1s activation by heat-aggregated IgG decreased at therapeutic hypothermia temperatures consistent with decreased enzymatic activity at lower temperatures. However, C1q binding to antibody-coated erythrocytes increased at lower temperatures, suggesting that increased classical complement pathway activation is mediated by increased C1 binding at therapeutic hypothermia temperatures. PIC1 inhibited hypothermia-enhanced complement-mediated cell lysis at 31°C by up to 60% (P = 0.001) in a dose dependent manner.
In summary, therapeutic hypothermia temperatures increased antibody-initiated complement activation and eukaryotic cell destruction suggesting that the benefits of therapeutic hypothermia may be mediated via other mechanisms. Antibody-initiated complement activation has been shown to contribute to ischemia-reperfusion injury in several animal models, suggesting that for diseases with this mechanism hypothermia-enhanced complement activation may partially attenuate the benefits of therapeutic hypothermia.
Available from: Muhammad Munir
- "It has been demonstrated that the capsid protein of human astrovirus type 1 blocks the classical  and lectin  activation pathways of the complement system, resulting in suppression of the downstream complement activation products and inflammatory mediators , , , . The region involved in inhibition of complement activation has been mapped to span amino acids 79–139 , . The CPΔN protein, where the truncation spans amino acids 1–160 presumably can overcome suppression of the immune system that has been described for the capsid protein. "
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ABSTRACT: Astroviruses are becoming a growing concern in veterinary and public health. To date there are no registered vaccines against astrovirus-induced disease, mostly due to the difficulty to cultivate astroviruses to high titer for vaccine development using conventional techniques. As means to circumvent this drawback, we have developed stably transfected mink fetal cells and BHK21 cells constitutively expressing the full-length and truncated capsid proteins of two distinct genotypes of mink astrovirus. Protein expression in these stably transfected cells was demonstrated by strong signals as evaluated by in-situ PLA and IFA, and confirmed by Western blotting. The recombinant full-length and truncated proteins induced a high level of antibodies in mink, evaluated by ELISA, demonstrating their immunogenicity. In a challenge experiment in mink, a reduction in presentation clinical signs and virus shedding was observed in mink kits born from immunized females. The gene integration and protein expression were sustained through cell passage, showing that the used approach is robust and reliable for expression of functional capsid proteins for vaccine and diagnostic applications.
Available from: Ernesto T.A. Marques
- "As expected, DENV bound to the wells containing 2H2 antibody and C1q, but not BSA (Fig. 2). A structural protein of human astrovirus type 1, the coat protein (CP), has been demonstrated to bind C1q (Bonaparte et al., 2008), inhibiting activation of classical complement pathway in vitro (Hair et al., 2010). However, in similar conditions, ENV protein from all Fig. 3. Incubation of DENV2 with C1q leads to a decrease in infection and in the levels of transcription of cellular factors. "
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ABSTRACT: Dengue virus infection elicits a spectrum of clinical presentations ranging from asymptomatic to severe disease. The mechanisms leading to severe dengue are not known, however it has been reported that the complement system is hyper-activated in severe dengue. Screening of complement proteins demonstrated that C1q, a pattern recognition molecule, can bind directly to Dengue Virus Envelope protein and to whole Dengue Virus serotype 2. Incubation of Dengue Virus serotype 2 with C1q prior to infection of THP-1 cells led to decreased virus infectivity and modulation of mRNA expression of immunoregulatory molecules suggesting reduced inflammatory responses.
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