Identification of C3b-binding regions on herpes simplex virus type 2 glycoprotein C

Abstract

Glycoprotein C from herpes simplex viruses types 1 and 2 (gC-1 and gC-2) acts as a receptor for the C3b fragment of the third component of complement. Our goal is to identify domains on gC involved in C3b receptor activity. Here, we used in-frame linker-insertion mutagenesis of the cloned gene for gC-2 to identify regions of the protein involved in C3b binding. We constructed 41 mutants of gC-2, each having a single, double, or triple insertion of four amino acids at sites spread across the protein. A transient transfection assay was used to characterize the expressed mutant proteins. All of the proteins were expressed on the transfected cell surface, exhibited processing of N-linked oligosaccharides, and bound one or more monoclonal antibodies recognizing distinct antigenic sites on native gC-2. This suggested that each of the mutant proteins was folded into a native structure and that a loss of C3b binding by any of the mutants could be attributed to the disruption of a specific functional domain. When the panel of insertion mutants was assayed for C3b receptor activity, we identified three distinct regions that are important for C3b binding, since an insertion within those regions abolished C3b receptor activity. Region I was located between amino acids 102 and 107, region II was located between residues 222 and 279, and region III was located between residues 307 and 379. In addition, region III has some structural features similar to a conserved motif found in complement receptor 1, the human C3b receptor. Finally, blocking experiments indicated that gC-l and gC-2 bind to similar locations on the C3b molecule.

Kinetic Analysis of Glycoprotein C of Herpes Simplex Virus Types 1 and 2 Binding to Heparin, Heparan Sulfate, and Complement Component C3b

“…Herpes simplex virus (HSV) codes for at least 11 glycoproteins, most of which are found on the virion envelope as well as infected cell surfaces (Spear, 1993a). Although glycoprotein C (gC) is not essential for viral growth in vitro (Draper et al, 1984;Holland et al, 1984;Johnson et al, 1986;Zezulak and Spear, 1984), it is rarely absent in clinical isolates (Arvin et al, 1983;Hidaka et al, 1990) and is associated with two important functions: entry of virus into cells via binding to cell-surface heparan sulfate (HS) (Gerber et al, 1995;Herold et al, 1991Herold et al, , 1995Sears et al, 1991;Tal-Singer et al, 1995) and immune evasion by HSV via binding to the C3b fragment of the third component of complement (Eisenberg et al, 1987;Friedman et al, 1984Friedman et al, , 1986Friedman et al, , 1996Harris et al, 1986;Hung et al, 1992;Lubinski et al, 1999Lubinski et al, , 1998Seidel-Dugan et al, 1990), leading to down-regulation of the complement cascade (Fries et al, 1986).…”

Resistance of herpes simplex virus type 2 to neomycin maps to the N-terminal portion of glycoprotein C

“…The significance of this difference is not obvious, since polylysine and other polycations inhibit adsorption of HSV-1 but not HSV-2 (15,16). Glycosylation also differs toward the N-terminal ends of the two glycoproteins (30).…”

Complement and immunity to viruses

“…Mutational analyses have implicated 3 regions in gC-2 (residues 102-107, 111-179 and 307-379) and 4 regions in gC-1 (residues 124-137, 276-292, 339-366 and 223-246) in the interaction with C3 (37,38). Region IV in gC-1 does not have a counterpart in gC-2 but the other regions are similarly positioned.…”