Mechanisms Of Viral Fusion Proteins

Viruses are major pathogens in humans, and in the organisms with which we share this planet. The massive health and economic burden these agents impose has spurred a huge research effort to understand their most intimate details. One outcome of this effort has been the production, in many but certainly not all cases, of effective vaccines and therapies. - other consequence has been the realization that we can exploit viruses and put them to work on our behalf. Viruses are still seen to have the most - tential as vehicles for gene delivery and other therapeutic applications. However, their ability to exploit cellular functions to their own ends makes viruses not only highly effective pathogens but also exquisite experimental tools. Work with viruses underpins much of our current understanding of molecular cell biology and related fields. For membrane traffic in parti- lar, viruses have been crucial in providing insights into key cellular fu- tions and the molecular mechanisms underlying these events.

Viral Fusion Mechanisms presents the first comprehensive review on this exciting topic. The book focuses on molecular mechanisms rather than phenomonology and examines a wide range of viruses, including influenza, HIV, Sendai, SFV, Vaccinia, VSV, and RSV. Recent theoretical work on dissecting protein-mediated membrane fusion is discussed, and the most promising new technologies for elucidating mechanisms are highlighted. Viral Fusion Mechanisms is an essential reference for biophysicists, cell biologists, colloid chemists, immunologists, microbiologists, molecular biologists, and virologists.

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry, the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Furthermore, the native pre-fusion structural and conformational dynamics of these fusion machines remains unclear as the conventional structural approaches employed by structural biologists are not well suited for studying these dynamic protein machines on the viral surface. The objective of this dissertation is to characterize the complete mechanism of Influenza virus hemagglutinin (HA) fusion activation and membrane fusion, and to profile and characterize the structural and conformational dynamics of the HIV-1 Env fusion glycoprotein on the viral surface. In chapter 2 I use continuous labeling HDX-MS to characterize the structural dynamics and conformational homogeneity of the HIV-1 Env fusion glycoprotein on the surface of two distinct engineered and authentic viral vaccine platforms. By HDX-MS we observed significant amounts of non-native Env present in one vaccine platform, whereas all Env present in the other resembled trimeric Env in the closed conformation. In chapter 3, I use pulse labeling HDX-MS to characterize the mechanism of HA fusion activation and HA mediated membrane fusion in situ using whole infectious virions. Our data reveal how concurrent reorganizations at the HA1 receptor binding domain interface and HA2 fusion subunit produce a dynamic fusion intermediate ensemble in full-length HA. In contrast, the soluble HA ectodomain transitions directly to the post-fusion state with no observable intermediate. These data provide unprecedented insight into the structural mechanics of HA which has served as the prototypical class 1 viral fusion protein and informed our understanding about how all class 1 viral fusion proteins function. In chapter 4 I present developments and improvements on the HDX-MS workflows that will enable more complete characterizations of HA's mechanism and the structural and conformational dynamics of other class 1 viral fusion proteins. Together these works have dramatically furthered our understanding of the structural mechanics of class 1 fusion proteins and lay the foundation for future studies on influenza virus and other enveloped viruses and their membrane fusion machinery.

"Principles of Molecular Virology, Fourth Edition" provides an essential introduction to modern virology in a clear and concise manner. It is a highly enjoyable and readable text with numerous illustrations that enhance the reader's understanding of important principles. It contains new material on virus structure, virus evolution, zoonoses, bushmeat, SARS and bioterrorism. The standard version includes a CD-ROM with Flash animations, virtual interactive tutorials and experiments, self-assessment questions, useful online resources, along with the glossary, classification of subcellular infectious agents and history of virology.

Membrane fusion and targeting processes are tightly regulated and coordinated. Dozens of proteins, originating from both the cytoplasm and membranes are involved. The discovery of homologous proteins from yeast to neurons validates a unified view. Although much is known about the interfering proteins, the events occurring when two lipid bilayers actually fuse are less clear. It should be remembered that lipid bilayers behave like soap-bubbles fusing when meeting each other. In this respect interfering proteins should be considered as preventing undesirable and unnecessary fusion and eventually directing the biological membrane fusion process (when, where, how, and overcoming the activation energy). In this latest volume in the renowned Subcellular Biochemistry series, some aspects of fusion of biological membranes as well as related problems are presented. Although not complete, there is a lot of recent information including on virus-induced membrane fusion. The contributors of the chapters are all among the researchers who performed many of the pioneering studies in the field.

Membrane fusion is an essential step in various developmental, physiological, and pathological processes in eukaryotes. Specific fusion proteins catalyze the merger of two lipid bilayers. The molecular mechanisms of many membrane fusion reactions still remain unclear. Here, we characterized the molecular details of various adhesion and fusion proteins involved in viral-cell, placental trophoblast, and sperm-egg fusion events to better understand their mechanisms. In total, nine crystal structures of these human and viral glycoproteins are presented. Furthermore, structural comparisons combined with mutagenesis, biochemical, biophysical, and cell culture experiments allowed us to investigate the implications of specific residues of these proteins in mediating membrane fusion. Structures of class I viral fusion proteins revealed that electrostatic interactions are critical for post-fusion stability in viruses that fuse at neutral pH, whereas the salt bridges do not play a stabilizing role in fusion proteins that proceed through low pH. Instead, hydrophobic residues stabilize the fusion core, and histidine/arginine residues in the chain reversal region stabilize a helix-dipole moment. Moreover, the structure of human syncytin-1 fusion protein unveiled a striking overall structural similarity to class I viral fusion proteins, and the presence of salt bridges within the fusion subunit zipper the inner and outer helices. These electrostatic interactions stabilize the post-fusion structure and provide the energetics for trophoblast cell fusion in placentation. While class I fusion proteins share similar structural and functional features, the mechanism of sperm-egg fusion requires the involvement of sperm Izumo1 and egg Juno proteins. Atomic resolution structures of Izumo1 and Juno display an interface stabilized through extensive interactions and a major conformational change within Izumo1 upon Juno binding. Moreover, mutational studies at the Izumo1-Juno interface revealed the structural determinants required for binding. We provide a comprehensive analysis that now identifies general trends and signatures important in viral-cell and cell-cell fusion events.

The publication of this volume of The Viruses entitled The Togaviridae and Flaviviridae comes at an appropriate time. The structure and rep lication strategies of these viruses are now known to be sufficiently di verse to warrant the removal of flaviviruses from the Togaviridae family and establish them as an independent family. Flaviviridae have a special place in the history of virology. The prototype virus-yellow fever virus was the first virus to be identified as the cause of a human disease. Some of the history of this discovery is described in Chapter 1 of this volume; in Chapter 10 the complete sequence of the RNA genome of the virus is presented. This sequence not only defines the primary structure of the viral proteins, it also clarifies the mechanism of translation of the fla vivirus genome. Knowledge of the sequence of the structural proteins of these viruses represents an important step in the potential goal of using purified flavivirus glycoproteins as vaccines. Many of the chapters in this volume focus on the structure and replication of the Togaviridae. These viruses have provided valuable models for studies in cell biology, partic ularly with regard to the cotranslational and posttranslational steps re quired for the synthesis and localization of membrane glycoproteins. Fur thermore, Togaviridae have been pivotal in our growing understanding of how enveloped viruses enter and exit from cells. The broad outlines of the structure and gene expression of Togavir idae and Flaviviridae are known, but important questions remain.

In this new edition of The Membranes of Cells, all of the chapters have been updated, some have been completely rewritten, and a new chapter on receptors has been added. The book has been designed to provide both the student and researcher with a synthesis of information from a number of scientific disciplines to create a comprehensive view of the structure and function of the membranes of cells. The topics are treated in sufficient depth to provide an entry point to the more detailed literature needed by the researcher. Key Features * Introduces biologists to membrane structure and physical chemistry * Introduces biophysicists to biological membrane function * Provides a comprehensive view of cell membranes to students, either as a necessary background for other specialized disciplines or as an entry into the field of biological membrane research * Clarifies ambiguities in the field