Interactions between herpes simplex virus type 1 glycoprotein C and cell surface glycosaminoglycans

Abstract

The cell surface glycosaminoglycan (GAG) heparan sulfate (HS) serves as an initial receptor for herpes simplex virus type 1 (HSV-1) and virus attachment to the HS molecule is mediated by the envelope glycoprotein C (gC). In the first part of this thesis, we aimed to define the HS-binding domain of viral gC. Virus mutants were constructed by site directed mutagenesis and a total of 14 mutant strains carrying single or dual amino acid substitutions in gC were available for functional characterization. These studies mapped the HS-binding site to a cluster of positively charged and hydrophobic/aromatic amino acids delimited by residues 129-160 in the antigenic site II of gC that had significant impact on the attachment and infectivity of the virus. HSV-1 utilizes the GAG chondroitin sulfate (CS) as an alternative or complementary receptor. We identified gC as the structure mediating viral attachment to CS. The positively charged and hydrophobic/aromatic amino acids essential for HS-binding were also critical for attachment to CS. The HS and CS binding domains of gC, although overlapping, were not identical. The interactions between gC and these two GAG molecules exhibited subtle but distinct differences in resistance to ionic strength, as well as to heparin and desulfated variants of this molecule. The second part of the study was designed to characterize an HSV-1 type-specific epitope of gC which is recognized by the monoclonal antibody (MAb), B1C1. This MAb is capable of inhibiting viral attachment. The studies revealed that the positively charged residues Arg143 to Arg155 within the HS-binding domain of gC were also essential for binding to B1C1. Furthermore, Thr150 within the 148NST150 N-glycosylation site was identified as a key residue for the epitope, but did not contribute to HS binding. In contrast, the complex-type N-glycan, shown to be present at this position in wild-type gC, did not influence binding to MAb B1C1 or HS. These studies demonstrated that the epitope of B1C1 partly overlapped the GAG binding domain of gC.In the last part of the thesis, we investigated whether MAb B1C1, and a synthetic peptide overlapping part of the GAG binding domain, inhibited attachment and infectivity of HSV-1 by interfering with the function of gC during viral entry. The MAb B1C1 neutralized viral infectivity efficiently, and blocked virus attachment to cell surfaces that expose either HS or CS. Human antibodies to gC displayed similar inhibitory effects. The peptide also showed antiviral activities in the form of inhibition of attachment and infectivity. These properties were dependent on the positively charged and hydrophobic amino acids previously demonstrated to be essential for HS binding of gC. Therefore, blocking of the functional domain of gC had profound consequences for HSV-1 infection in the cell culture systems studied. The demonstration of mechanisms for interference with virus infection through inhibition of attachment of HSV-1 by antibodies and synthetic peptides may be of value for the development of new antiviral strategies against herpesviruses.