Herpes Simplex Virus Type 2 Mucin-Like Glycoprotein mgG Promotes Virus Release from the Surface of Infected Cells.

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

Regulatory Mechanisms of the Mucin-Like Region on Herpes Simplex Virus during Cellular Attachment.

Mucin-like regions, characterized by a local high density of O-linked glycosylation, are found on the viral envelope glycoproteins of many viruses. Herpes simplex virus type 1 (HSV-1), for example, exhibits a mucin-like region on its glycoprotein gC, a viral protein involved in initial recruitment of the virus to the cell surface via interaction with sulfated glycosaminoglycans. So far, this mucin-like region has been proposed to play a key role in modulating the interactions with cellular glycosaminoglycans, and in particular to promote release of HSV-1 virions from infected cells. However, the molecular mechanisms and the role as a pathogenicity factor remains unclear. Using single virus particle tracking, we show that the mobility of chondroitin sulfate-bound HSV-1 virions is decreased in absence of the mucin-like region. This decrease in mobility correlates with an increase in HSV-1-chondroitin sulfate binding forces as observed using atomic force microscopy-based force spectroscopy. Our data suggest that the mucin-like region modulates virus-glycosaminoglycan interactions by regulating the affinity, type, and number of glycoproteins involved in the virus-glycosaminoglycan interaction. This study therefore presents new evidence for a role of the mucin-like region in balancing the interaction of HSV-1 with glycosaminoglycans and provides further insights into the molecular mechanisms used by the virus to ensure both successful cell entry and release from the infected cell.

Cell Membrane Derived Platform To Study Virus Binding Kinetics and Diffusion with Single Particle Sensitivity.

Discovery and development of new antiviral therapies essentially rely on two key factors: an in-depth understanding of the mechanisms involved in viral infection and the development of fast and versatile drug screening platforms. To meet those demands, we present a biosensing platform to probe virus-cell membrane interactions on a single particle level. Our method is based on the formation of supported lipid bilayers from cell membrane material. Using total internal reflection fluorescence microscopy, we report the contribution of viral and cellular components to the interaction kinetics of herpes simplex virus type 1 with the cell membrane. Deletion of glycoprotein C (gC), the main viral attachment glycoprotein, or deletion of heparan sulfate, an attachment factor on the cell membrane, leads to an overall decrease in association of virions to the membrane and faster dissociation from the membrane. In addition to this, we perform binding inhibition studies using the antiviral compound heparin to estimate its IC50 value. Finally, single particle tracking is used to characterize the diffusive behavior of the virus particles on the supported lipid bilayers. Altogether, our results promote this platform as a complement to existing bioanalytical assays, being at the interface between simplified artificial membrane models and live cell experiments.

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans.

Many viruses, including herpes simplex (HSV), are recruited to their host cells via interaction between their envelope glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial attachment is of a multivalent nature, i.e., it requires the establishment of multiple bonds between amino acids of viral glycoproteins and sulfated saccharides on the GAG chain. To gain understanding of how this binding process is modulated, we performed binding kinetics and mobility studies using end-grafted GAG chains that mimic the end attachment of these chains to proteoglycans. Total internal reflection fluorescence microscopy was used to probe binding and release, as well as the diffusion of single HSV-1 particles. To verify the hypothesis that the degree of sulfation, but also the arrangement of sulfate groups along the GAG chain, plays a key role in HSV binding, we tested two native GAGs (chondroitin sulfate and heparan sulfate) and compared our results to chemically sulfated hyaluronan. HSV-1 recognized all sulfated GAGs, but not the nonsulfated hyaluronan, indicating that binding is specific to the presence of sulfate groups. Furthermore we observed that a notable fraction of GAG-bound virions exhibit lateral mobility, although the multivalent binding to the immobilized GAG brushes ensures firm virus attachment to the interface. Diffusion was faster on the two native GAGs, one of which, chondroitin sulfate, was also characterized by the highest association rate per GAG chain. This highlights the complexity of multivalent virus-GAG interactions and suggests that the spatial arrangement of sulfates along native GAG chains may play a role in modulating the characteristics of the HSV-GAG interaction. Altogether, these results, obtained with a minimal and well-controlled model of the cell membrane, provide, to our knowledge, new insights into the dynamics of the HSV-GAG interaction.

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

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.