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. 2003 Mar;77(6):3878-81.
doi: 10.1128/jvi.77.6.3878-3881.2003.

Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Open Reading Frame 4 Protein (Kaposica) Is a Functional Homolog of Complement Control Proteins

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Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Open Reading Frame 4 Protein (Kaposica) Is a Functional Homolog of Complement Control Proteins

Jayati Mullick et al. J Virol. .
Free PMC article

Abstract

The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.

Figures

FIG. 1.
FIG. 1.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified ORF 4 protein (kaposica). Culture supernatant from an ORF 4 protein-expressing clone and the purified ORF 4 protein were run on a sodium dodecyl sulfate-10% polyacrylamide gel under reducing conditions and stained with Coomassie blue. Lane 1, molecular weight (MW) markers (numbers in kilodaltons); lane 2, culture supernatant; lane 3, purified kaposica.
FIG. 2.
FIG. 2.
Inhibition of alternative and classical pathway-mediated lysis of erythrocytes by kaposica and VCP. The relative effects of kaposica and VCP on the alternative pathway (AP)-mediated lysis of rabbit erythrocytes and the classical pathway (CP)-mediated lysis of antibody-coated sheep erythrocytes were examined as previously described (19). Bovine serum albumin (BSA) was included as a nonspecific control. IC50, 50% inhibitory concentration.
FIG. 3.
FIG. 3.
Inhibition of C3b deposition on erythrocytes during complement activation by kaposica and VCP. Classical pathway-mediated C3b deposition on erythrocytes was measured by incubating 5 μl of antibody-coated sheep erythrocytes (109/ml in GVB++: 5 mM barbital, 145 mM NaCl, 0.5 mM MgCl2, 0.15 mM CaCl2, and 0.1% gelatin, pH 7.4) with 1 μl of C8-deficient human serum (Calbiochem, San Diego, Calif.) and 44 μl of GVB++ or GVB++ containing 2 μM kaposica or VCP at 37°C for 30 min. Deposition of C3b was detected by fluorescence-activated cell sorting with fluorescein isothiocyanate-conjugated F(ab′)2 anti-C3 goat immunoglobulin G (Cappel Laboratories, Warrington, Pa.). Control samples contained either 10 mM EDTA or 2 μM bovine serum albumin (BSA).
FIG. 4.
FIG. 4.
Analysis of factor I cofactor activity of kaposica and VCP for complement proteins C3b (upper panel) and C4b (lower panel). Cofactor activity was observed by incubating C3b or C4b (4 μg) with kaposica (2 μg) or VCP (2 μg) and factor I (100 ng) in 15 μl of 10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl at 37°C for the indicated time period. Cleavage products were visualized by running the samples on sodium dodecyl sulfate-8 and 9% polyacrylamide gels for C3b and C4b, respectively, and staining them with Coomassie blue. During C3b cleavage, the α′ chain is cleaved into N-terminal 68-kDa and C-terminal 43-kDa fragments; the appearance of these fragments indicates the generation of iC3b (inactive C3b). In the case of C4b cleavage, the α′ chain is cleaved into N-terminal 25-kDa, C-terminal 16-kDa, and central C4d fragments; these cleavages result in inactivation of C4b and generation of C4c and C4d.

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