Antagonism of the complement component C4 by flavivirus nonstructural protein NS1

The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural 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 virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although 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 degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection

Analysis of the putative role of CR1 in Alzheimer’s disease: Genetic association, expression and function

Chronic activation of the complement system and induced inflammation are associated with neuropathology in Alzheimer's disease (AD). Recent large genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the C3b/C4b receptor (CR1 or CD35) that are associated with late onset AD. Here, anti-CR1 antibodies (Abs) directed against different epitopes of the receptor, were used to localize CR1 in brain, and relative binding affinities of the CR1 ligands, C1q and C3b, were assessed by ELISA. Most Abs tested stained red blood cells in blood vessels but showed no staining in brain parenchyma. However, two monoclonal anti-CR1 Abs labeled astrocytes in all of the cases tested, and this reactivity was preabsorbed by purified recombinant human CR1. Human brain-derived astrocyte cultures were also reactive with both mAbs. The amount of astrocyte staining varied among the samples, but no consistent difference was conferred by diagnosis or the GWAS-identified SNPs rs4844609 or rs6656401. Plasma levels of soluble CR1 did not correlate with diagnosis but a slight increase was observed with rs4844609 and rs6656401 SNP. There was also a modest but statistically significant increase in relative binding activity of C1q to CR1 with the rs4844609 SNP compared to CR1 without the SNP, and of C3b to CR1 in the CR1 genotypes containing the rs6656401 SNP (also associated with the larger isoform of CR1) regardless of clinical diagnosis. These results suggest that it is unlikely that astrocyte CR1 expression levels or C1q or C3b binding activity are the cause of the GWAS identified association of CR1 variants with AD. Further careful functional studies are needed to determine if the variant-dictated number of CR1 expressed on red blood cells contributes to the role of this receptor in the progression of AD, or if another mechanism is involved

Binding of Flavivirus Nonstructural Protein NS1 to C4b Binding Protein Modulates Complement Activation

The complement system plays a pivotal protective role in the innate immune response to many pathogens including flaviviruses. Flavivirus nonstructural protein 1 (NS1) is a secreted nonstructural glycoprotein that accumulates in plasma to high levels and is displayed on the surface of infected cells but absent from viral particles. Previous work has defined an immune evasion role of flavivirus NS1 in limiting complement activation by forming a complex with C1s and C4 to promote cleavage of C4 to C4b. In this study, we demonstrate a second mechanism, also involving C4 and its active fragment C4b, by which NS1 antagonizes complement activation. Dengue, West Nile, or yellow fever virus NS1 directly associated with C4b binding protein (C4BP), a complement regulatory plasma protein that attenuates the classical and lectin pathways. Soluble NS1 recruited C4BP to inactivate C4b in solution and on the plasma membrane. Mapping studies revealed that the interaction sites of NS1 on C4BP partially overlap with the C4b binding sites. Together, these studies further define the immune evasion potential of NS1 in reducing the functional capacity of C4 in complement activation and control of flavivirus infection. The Journal of Immunology, 2011, 187: 424-433

Representation of CR1 structure, ligand binding sites and location of antibody epitopes.

<p>CYT: cytoplasmic domain, TM: transmembrane domain. Red dot indicates location of rs4844609 (Ser1610Thr). Diagram modified from[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149792#pone.0149792.ref016" target="_blank">16</a>]; Antibody reactivity modified from[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149792#pone.0149792.ref017" target="_blank">17</a>].</p

Case Characteristics for Analyzed Brain Samples.

<p>Case Characteristics for Analyzed Brain Samples.</p

CR1 expressed by CHO cells is recognized by anti-CR1 antibodies.

<p>CR1 immunofluorescence staining using mouse mAbs 8C9.1(A), E11 (B) or J3B11 (C) on fixed CHO cells expressing 1x10⁶ (high) (upper panels) or 1x10⁵ (moderate) (middle panels) receptors/cell. Bottom panels show isotype control on high CHO-CR1 (A), or antibody reactivity on non-transfected CHO cells (B, C). Scale bar: 50 μm.</p

Electrophoretic mobility (Mr) and gene frequency of the allelic size isoforms of CR1.

<p>Electrophoretic mobility (Mr) and gene frequency of the allelic size isoforms of CR1.</p

CR1/CD35 expression in human brain.

<p>Representative micrographs of brain sections stained with monoclonal anti-CR1 Abs 8C9.1 (A, B, D-F), J3B11 (G-I) or mIgG control (C) in frontal cortex of control (CTR) and Alzheimer’s disease (AD) cases. Genotype for the two GWAS SNPs rs4844609 (T or A) and rs6656401 (G or A) are presented in each panel. Scale bar 100 μm.</p

Anti-CR1 immunoreactivity with astrocytes but not neurons is preabsorbed by rhCR1.

<p>Immunostaining with monoclonal anti-CR1 8C9.1 (A, B) or J3B11 (C) without (upper panels) or with (lower panels) preabsorption with recombinant human CR1. Neuronal staining (B) was not preabsorbed with rhCR1. Scale bar: A, C: 50 μm, B: 100 μm.</p