Regarding to hypothesis3, the model starts with the fusion loops pointing toward the viral membrane with an intermediate-containing compact form with the fusion loops pointing up. nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to place fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for any neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gBs conformational BMS-214662 rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral access and therefore infectivity. KEYWORDS:cryo-electron microscopy, cryo-electron tomography, HSV, subtomogram averaging, gB, herpesviruses, microvesicles, neutralizing antibodies, prefusion, viral fusion == IMPORTANCE == The BMS-214662 herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse Rabbit Polyclonal to PPIF their envelope with the host membrane to start an infection. This process is usually mediated by a viral surface protein that transitions from an initial conformation (prefusion) to a final, more stable, conformation (postfusion). However, the prefusion conformation of the herpesvirus fusion protein (gB) is usually poorly comprehended. To elucidate the structure of the prefusion conformation of HSV type 1 gB, we have employed cryo-electron microscopy to study gB molecules expressed on the surface of vesicles. Using different approaches to label gBs domains allowed us to model the structures of the prefusion and intermediate conformations of gB. Overall, our findings enhance our understanding of HSV fusion and lay the groundwork for the development of new ways to prevent and block HSV contamination. == INTRODUCTION == Herpes simplex virus (HSV) is usually a model system for the herpesvirus family, which includes human viruses that cause lifelong infections and a variety of diseases, including skin lesions, encephalitis, and cancers. HSV, which is usually categorized into two types (HSV-1 and HSV-2), also causes a highly contagious contamination common and endemic throughout the world. It is estimated that over 3.5 billion people worldwide are infected with HSV-1, while over 400 million people are infected with HSV-2, an infection that has been shown to increase the risk of HIV acquisition (1). Antivirals that reduce the severity and frequency of HSV symptoms exist. However, these drugs cannot cure contamination and there is no HSV vaccine available. A key step of viral contamination is usually entry into the host cell, a process that for enveloped viruses like HSV entails fusion of viral and cellular membranes, allowing the viral genome to access the interior of the cell. Enveloped computer virus fusion is usually mediated by viral transmembrane proteins, and mounting evidence suggests that these proteins have converged on a similar overall strategy among different viruses and classes of fusion proteins (2). Herpesvirus access and membrane fusion require three virion glycoproteins that function as the core fusion machinery, gB, the actual fusion protein, and the gH/gL heterodimer (3). Additionally, HSV fusion requires the gD glycoprotein (4). Atomic models for many of the HSV glycoproteins exist, including for gD in its unliganded form BMS-214662 (5) and in complex with its receptors (69); for any partially activated gH/gL complex (10); and for the postfusion form of gB (11,12). Structures of Epstein-Barr computer virus gH/gL alone and in complex with gp42 (13,14) and gB (15), human cytomegalovirus gB (16,17), pseudorabies computer virus gH/gL (18), and varicella-zoster computer virus gH/gL (19) are also available. Current HSV fusion models propose that receptor-activated gD converts the regulatory protein gH/gL to an active state, which in turn promotes the fusogenic ability of gB, the fusion protein (20). A detailed description of this process is usually reviewed elsewhere (4,21). Of notice, Rogalin and Heldwein have recently generated vesicular stomatitis computer virus (VSV) particles pseudotyped with HSV-1 gD, gH/gL, and gB, and these particles were found to be able to.
Regarding to hypothesis3, the model starts with the fusion loops pointing toward the viral membrane with an intermediate-containing compact form with the fusion loops pointing up
