Virus-cell membrane interactions - Binding studies of Herpes Simplex Virus using surface-sensitive techniques

Sammanfattning: Viruses are parasites capable of infecting all forms of life. They lack the ability to replicate by themselves and therefore hijack the replication machinery of cells to produce new viral copies, called virions. Two steps of this replication cycle are of particular interest in the frame of this thesis: the initial attachment of the virus to the cell membrane and the release of the virus from the cell membrane after infection. One virus for which these mechanisms are still under investigation is the herpes simplex virus (HSV). HSV is commonly known for causing blisters on the skin or mucosa of the lips, mouth or genitals. In rare cases, it can migrate to the central nervous system, causing meningitis or encephalitis. HSV is an enveloped DNA virus that binds to the cell membrane via interactions between viral glycoproteins and cell-surface sulfated polysaccharide chains, called glycosaminoglycans (GAGs). The main focus of the work presented here was the interaction between HSV and surface immobilized GAGs. The individual GAG chains were end-grafted to a sensor surface to mimic the brush-like architecture of GAGs found close to the cell surface. Total internal reflection fluorescence microscopy (TIRFM) was used to extract information on binding kinetics and mobility of single fluorescently labeled HSV particles. Two aspects of the HSV-GAG interaction were studied in detail: First, the influence of the sulfation of the GAG chains on the binding characteristics, and second, the role of glycosylation of the viral glycoproteins. Binding studies of HSV to different GAGs showed that the degree of sulfation of the GAG influences the binding affinity of HSV. Furthermore, single particle tracking (SPT) analysis revealed that HSV diffuses on the GAG surface and that this mobility is influenced by the affinity of the HSV-GAG bonds. Finally, experiments involving virus mutants demonstrated that the glycosylation of the viral glycoproteins plays a critical role in the release of HSV virions from the surface of infected cells. Taken together, two aspects modulating the interaction between the herpes simplex virus and glycosaminolgycans were identified in this thesis, providing new insights into the mechanisms that regulate the initial attachment of the virus to the cell membrane and its release from the cell membrane after infection. In addition, the mobility of the HSV particles observed with the model system suggests that the virus is able to diffuse on the cell membrane in vivo.