Mechanism of complement inactivation by glycoprotein C of herpes simplex virus.

Glycoprotein C (gC) of both herpes simplex virus type 1 (HSV-1) and HSV-2 interacts with complement C3b and protects the virus from complement-mediated neutralization. To study the mechanism by which gC modulates complement activation, we expressed both gC-1 and gC-2 in a baculovirus expression system. Baculovirus recombinants containing gC genes spanning the entire gC-1 sequence (gC-1-TMR) or only the extracellular domain(s) of gC-1, gC-2, or a deletion mutant of gC-1 lacking residues 33 through 123 were expressed in sf9 insect cells. Binding of the expressed proteins to human C3 and C3 fragments was assessed by direct and competition ELISA. All four expressed proteins bound to C3, C3b, and C3c but not to C3d, suggesting 1) that the binding sites for these proteins are located in the C3c region of C3; and 2) that gC, in contrast to other C3-binding proteins, interacts with native C3. We have also examined the interaction of native C3 with gC-1 expressed on the HSV-1-infected cells. Analogous to recombinant proteins, gC-1 expressed on the infected cells also bound to native C3. The ability of baculovirus-expressed gCs to inhibit the interaction of C3b with its ligands was also analyzed. We found that gC-1, but not gC-2, inhibited the binding of C5 and properdin to C3b and also inhibited the alternative pathway-mediated lysis of rabbit erythrocytes. Inhibition of alternative pathway-mediated lysis and properdin binding to C3b, but not of C5 binding to C3b, required the transmembrane segment of the gC-1. The specificity of gC interactions was examined by studying the interaction of gC with C3 from various species. In contrast to properdin, both gCs bound to cobra C3; this finding suggests that gC-1 and properdin bind to different sites on C3b. Further analyses suggested that gC-1 sterically hindered access of C5 and properdin to C3b.

Overexpression, purification, and characterization of third component of complement.

The third component of complement (C3) plays a critical role in both pathways of complement activation by interacting with numerous other complement proteins. To elucidate the molecular features of C3 that relate to the functional activities of the molecule, we expressed the cDNA of human complement component C3 in cultured insect cells using a baculovirus expression vector system derived from the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). The expression of C3 was controlled by the promoter of the polyhedrin gene and, when recombinant baculovirus infected insect cells were cultured in serum-free medium, C3 was detected at a level of 10 micrograms/ml of culture medium. Characterization of the recombinant C3 (rC3) by SDS-PAGE revealed that the C3 gene product was translated as a 188 kDa protein comprised of two chains of 115 kDa and 73 kDa analogous to the alpha and beta chains of serum-derived human C3 (sC3). An analysis of the glycosylation pattern of purified rC3 revealed that, whereas both the alpha and beta chains were glycosylated as in sC3, the proC3 moiety of rC3 also was glycosylated. When rC3 was produced in the High Five cell line of insect cells and evaluated for reactivity with a panel of anti-C3 monoclonal antibodies (MoAb), the results suggested that the conformation of the baculovirus expressed C3 was similar to that of native C3. When the rC3 was purified by anion exchange column chromatography, it was able to react with several C3-binding proteins (CR1, P and H), reconstitute C3-deficient serum and support the activation of both complement pathways thus demonstrating that a baculovirus-expressed C3 can participate in the formation of and can be cleaved by both the classical and alternative pathway convertases. Incubation of rC3 with factor I and H revealed that both C3 and proC3 are susceptible to cleavage by factor I.

The interaction of glycoprotein C of herpes simplex virus types 1 and 2 with the alternative complement pathway.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) binds the human complement fragment C3b, but the two proteins differ in their ability to bind C3b on infected cell surfaces. In addition, gC-1, but not gC-2, accelerates the decay of the alternative pathway C3 convertase, thereby affecting later steps of the complement cascade. Previously, we constructed linker insertion and deletion mutants of gC-1 and gC-2 and used transient transfection to express mutant proteins in uninfected cells. In spite of the differences between gC-1 and gC-2, C3b binding was localized to residues within the central portion of both proteins, encompassing the first four cysteines. For gC-1, deletion mutants lacking amino acids 33 to 123 or 367 to 469 or lacking both regions still bound C3b. We recombined these deleted forms of gC-1 into gC-39, an HSV-1 strain lacking the gC gene. The altered forms of gC-1 were incorporated into virions, expressed on the surface of infected cells, and bound C3b. We used these proteins to investigate the structural basis for the inhibitory action of gC-1 on the complement cascade. We found that gC-1 does not inhibit formation of the alternative pathway C3 convertase. This convertase is stabilized by the serum protein properdin. Purified gC-1, but not gC-2, inhibits the binding of properdin to C3b, suggesting that this destabilizes the convertase. The mutant lacking amino acids 367 to 449 was able to inhibit properdin binding to a limited extent when present at high concentrations, although it bound to C3b more weakly than wild-type gC. In contrast, the protein lacking amino acids 33 to 123 was unable to inhibit properdin binding to C3b. Thus, gC-1 contains two structural domains, one for C3b binding, residues 124 to 366, and another, residues 33 to 133, which interferes with properdin binding to C3b.