Search

The molecular mechanism of entry of herpesviruses requires a multicomponent fusion system. Cell invasion by Herpes simplex virus (HSV) requires four virally encoded glycoproteins: namely gD, gB and gH/gL. The role of gB has remained elusive until recently when the crystal structure of HSV-1 gB became available and the fusion potential of gB was clearly demonstrated. Although much information on gB structure/function relationship has been gathered in recent years, the elucidation of the nature of the fine interactions between gB fusion loops and the membrane bilayer may help to understand the precise molecular mechanism behind herpesvirus-host cell membrane fusion. Here, we report the first biophysical study on the two fusion peptides of gB, with a particular focus on the effects determined by both peptides on lipid bilayers of various compositions. The two fusion loops constitute a structural subdomain wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. When used together the two fusion loops have the ability to significantly destabilize the target membrane bilayer, notwithstanding their low bilayer penetration when used separately. These data support the model of gB fusion loops insertion into cholesterol enriched membranes.

Given the specific melanin-associated paramagnetic features, the Electron Spin Resonance (ESR, called also Electron Paramagnetic Resonance, EPR) analysis has been proposed as a potential tool for non-invasive melanoma diagnosis. However, studies comparing human melanoma tissues to the most appropriate physiological counterpart (nevi) have not been performed, and ESR direct correlation with melanoma clinical features has never been investigated. ESR spectrum was obtained from melanoma and non-melanoma cell-cultures as well as mouse melanoma and non-melanoma tissues and an endogenous ESR signal (g = 2.005) was found in human melanoma cells and in primary melanoma tissues explanted from mice, while it was always absent in non-melanoma samples. These characteristics of the measured ESR signal strongly suggested its connection with melanin. Quantitative analyses were then performed on paraffin-embedded human melanoma and nevus sections, and validated on an independent larger validation set, for a total of 112 sections (52 melanomas, 60 nevi). The ESR signal was significantly higher in melanomas (p=0.0002) and was significantly different between ‘‘Low Breslow’s and ‘‘High Breslow’s’’ depth melanomas (p,0.0001). A direct correlation between ESR signal and Breslow’s depth, expressed in millimetres, was found (R=0.57; p,0.0001). The eu/pheomelanin ratio was found to be significantly different in melanomas ‘‘Low Breslow’s’’ vs melanomas ‘‘High Breslow’s’’ depth and in nevi vs melanomas ‘‘High Breslow’s depth’’. Finally, ROC analysis using ESR data discriminated melanomas sections from nevi sections with up to 90% accuracy and p,0.0002. In the present study we report for the first time that ESR signal in human paraffin-embedded nevi is significantly lower than signal in human melanomas suggesting that spectrum variations may be related to qualitative melanin differences specifically occurring in melanoma cells. We therefore conclude that this ESR signal may represent a reliable marker for melanoma diagnosis in human histological sections.