Molecular basis of the glycoprotein C-negative phenotypes of herpes simplex virus type 1 mutants selected with a virus-neutralizing monoclonal antibody

Abstract

we described the isolation and partial characterization of over 100 herpes simplex virus type 1 mutants which were resistant to neutralization by a pool of glycoprotein C-(gC) specific monoclonal antibodies. The genetic basis for the inability of several of these gC mutants to express an immunoreactive envelope form of gC is reported here. Comparative nucleotide sequence analysis of the gC gene of the six mutants gC-3, gC-8, gC-49, gC-53, gC-85, and synLD70, which secrete truncated gC polypeptides, with that of the wild-type KOS 321 gC gene revealed that these mutant phenotypes were caused by frameshift or nonsense mutations, resulting in premature termination of gC translation. Secretion of the gC polypeptide from cells infected with these mutants was due to the lack of a functional transmembrane anchor sequence. The six secretor mutants were tested for suppression of amber mutations in mixed infection with a simian virus 40 amber suppressor vector. Mutant gC-85 was suppressed and produced a wild-type-sized membrane-bound gC. Nucleotide sequence analysis of the six gC deletion mutants gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98 revealed that they carried identical deletions which removed 1,702 base pairs of the gC gene. The deletion, which was internal to the gC gene, removed the entire gC coding sequence and accounted for the novel 1.1-kilobase mRNA previously seen in infections with these mutants. The mutant gC-44 was previously shown to produce a membrane-bound gC protein indistinguishable in molecular weight from wild-type gC. This mutant differed from wild-type virus in that it had reduced reactivity with virus-neutralizing monoclonal antibodies. Nucleotide sequence analysis of the gC gene of mutant gC-44 demonstrated a point mutation which changed amino acid 329 of gC from a serine to a phenylalanine.

Localization on the herpes simplex virus type 1 genome of a region encoding proteins involved in adsorption to the cellular receptor

“…The intertypic HSV-1-HSV-2 recombinants used have been characterized earlier (40,51). The monoclonal-antibodyresistant gC mutants of HSV-1, the gC-39 mutant, and their parental strain HSV-1 KOS 321 have been described and characterized previously (23)(24)(25). The monoclonal antibodies 1001, 1105, and 2454 raised against gC have also been described previously (47).…”

“…Incubations were carried out in Eagle minimal essential medium containing 10% heat-inactivated serum. Routinely, 25 ,dl of ascitic fluid was used per ml when antibodies were incubated with virus; higher doses gave no further inhibition of adsorption. Binding of radiolabeled virus was measured similarly, except that after the adsorption period the cells were extensively washed and the remaining bound radioactivity was measured in a liquid scintillation counter.…”

Initial interaction of herpes simplex virus with cells is binding to heparan sulfate

“…Fuller, R. Santos, and P. G. Spear, manuscript in preparation). The other four known glycoproteins are apparently dispensable for viral replication in cultured cells (12,21,27,32). That gD and gB are present in different morphologically distinct spikes projecting from the virion envelope (38) implies a requirement for multiple virion-cell surface interactions subsequent to adsorption in order for virion-cell fusion to occur.…”

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

“…Glycoprotein C (gC) acts as a receptor for the C3b fragment of the third component of complement (18,21,45). Although gE, gI, and gC are not required for infection in cell culture (14,26,29,33,41,42,47,65), gE and gC are present in clinical isolates (2,22,52), suggesting that Fc and C3b receptor activities are important for viral pathogenesis in vivo.…”