Herpes Simplex Virus Type 2 Glycoprotein G Is Targeted by the Sulfated Oligo- and Polysaccharide Inhibitors of Virus Attachment to Cells

Abstract

Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gGdeficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo-and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans.It is well-established that cell surface heparan sulfate (HS) chains provide the binding sites for the initial interactions with cells of many viruses, including herpes simplex virus type 1 (HSV-1) and HSV-2 (38). The two types of HSV differ in their interactions with HS with respect to both the viral glycoproteins and the HS motifs involved. In particular, glycoprotein C (gC) of HSV-1 was identified as a component of the viral envelope that interacts with HS/heparin chains, thus mediating the attachment of the virus to cells (15). Although gC of HSV-2 can bind to HS/heparin chains and was found to be responsible for several HSV type-specific differences, such as polycation (28) and the hypertonic medium (36) resistance of HSV-2 infection of cells, this protein did not mediate HSV-2 attachment to cells (11). Instead, gB, another HS-binding component of the HSV envelope, was identified as the major virus attachment protein (5). In addition to gB and gC, gD of HSV-1, but not its HSV-2 homolog, can bind to HS chains modified by several isoforms of 3-O-sulfotransferase (31), an interaction that triggers HSV-1 entry into cells. Thus, interaction of HSV with HS seems to be a complex process that involves several kinds of viral proteins promoting virus attachment to and entry into cells.Compounds such as sulfa...

Glycoproteins gB and gH Are Required for Syncytium Formation but Not for Herpesvirus-Induced Nuclear Envelope Breakdown

“…The enhanced fusogenicity correlates with mutations either in gB (45,(47)(48)(49)(50), gK (46), or pUL20 (51). Syncytial mutations in gB were found in two conserved regions of the cytoplasmic domain.…”

“…Serial passage of herpesviruses often results in the increased formation of syncytia (45,46). The enhanced fusogenicity correlates with mutations either in gB (45,(47)(48)(49)(50), gK (46), or pUL20 (51).…”

The Cholestanol-Conjugated Sulfated Oligosaccharide PG545 Disrupts the Lipid Envelope of Herpes Simplex Virus Particles

“…Each fraction was subjected to (i) titration of residual infectivity in the viral plaque assay, (ii) quantification of radioactivity in a scintillation counter, (iii) determination of the number of viral DNA copies based on quantitative PCR (qPCR) analysis (35), and (iv) determination of reactivity with mouse monoclonal antibodies against HSV glycoprotein B (gB) (clone B11D8; dilution, 1:100), gC (clone E5F7; dilution, 1:100), gE (clone B1E6; dilution, 1:100) and gG (clone O1C5; dilution, 1:100) and rabbit polyclonal antibody against the major capsid protein VP5 (NC1; dilution, 1:500) by an enzyme-linked immunosorbent assay (ELISA)-based method. The monoclonal antibodies were prepared and characterized in our laboratory (26,36,37), while polyclonal antibody NC1 was kindly provided by Gary Cohen and Roselyn Eisenberg (University of Pennsylvania).…”

“…We have previously found that muparfostat (formerly known as PI-88), a mixture of highly sulfated oligosaccharides, inhibited infectivity of HSV (25)(26)(27), RSV (28), and HIV-1 (29) in cultured cells without causing permanent inactivation of viral infectivity. However, the cholestanol-conjugated sulfated tetrasaccharide of muparfostat, also referred to as 14/P3 (27)(28)(29), and a related compound known as PG545 (30,31) showed the capability to permanently inactivate the infectivity of HSV, RSV, and HIV-1 (27)(28)(29).…”

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction

“…Interestingly, similar selection experiments performed with HSV-2 resulted in viral variants lacking gG, a virus envelope protein also carrying a mucin-like region (13). These results, indicating that the mucin-like regions of viral glycoproteins could modify the virus sensitivity to GAG mimetics, encouraged us to further investigate the influence of these domains on the GAG-binding activity of viral attachment.…”