| 1. |
- Goodfellow, IG, et al.
(author)
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Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors
- 2005
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In: Journal of Virology. - 1098-5514. ; 79:18, s. 12016-12024
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Journal article (peer-reviewed)abstract
- We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.
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| 2. |
- Mark, Linda, et al.
(author)
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Localization of functional sites in the viral complement inhibitor KCP
- 2004
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In: Immunology 2004: Cytokine Network, Regulatory Cells, Signaling, and Apoptosis. ; , s. 243-247
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Conference paper (peer-reviewed)abstract
- Kaposi's sarcoma-associated herpes virus (KSHV) encodes a complement inhibitor named KSHV complement control protein (KCP). We have previously shown that KCP inhibits the human complement system by disrupting the C3 convertase of complement. KCP can accelerate the decay of the classical C3 convertase and it can act as a cofactor for factor I (R), which then cleaves and inactivates C4b or C3b (in the classical and alternative convertase, respectively). The aim of this study was to 1) delineate the sites on KCP responsible for complement inhibition 2) study the binding of KCP to heparin and the surface of cells. We constructed a 3D model of the four CCP domains KCP by homology based modeling, which served as basis for site directed mutagenesis. A patch of positively charged amino acids in CCPI, stretching into CCP2 as well as a positive and a negative patch in the region between CCP2-3 were the most functionally important sites. KCP binds to heparin and the surface of cells via a site in CCPI.
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| 3. |
- Spiller, OB, et al.
(author)
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Dissecting the regions of virion-associated Kaposi's sarcoma-associated herpesvirus complement control protein required for complement regulation and cell binding
- 2006
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In: Journal of Virology. - 1098-5514. ; 80:8, s. 4068-4078
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Journal article (peer-reviewed)abstract
- Complement, which bridges innate and adaptive immune responses as well as Immoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not CA cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.
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