The matrix (M) protein is the major viral structural protein that regulates assembly and structure determination in members of the Para- and Ortho-myxoviridae families. The two families include numerous human pathogens such as human respiratory syncytial virus (hRSV), measles virus (MeV), human parainfluenza virus (hPIV), and the Influenza A, B, and C genera. The matrix protein initiates virus assembly by forming oligomeric sheets in specific regions along the host cell membrane. The M protein also orchestrates the incorporation of the viral glycoproteins and the ribonucleoprotein (RNP) into the evolving viral particles. Here we aim to determine two things: 1) the native structure and organization of RSV, which is an archetypical member of the Paramyxoviridae family;and 2) how the RSV M protein regulates virus assembly and determines virus morphology. To answer these questions, we are using the A2 strain of RSV that causes bronchiolitis, viral pneumonia, and death in young children, the elderly, and immuno-compromised individuals. We are analyzing RSV structure to build upon our previous structural studies of HIV-1 assembly and maturation, our ongoing structural analysis of native measles virus glycoprotein arrangements and interactions, and to develop RSV as a model system for future structural studies of virus assembly, virus organization, and the development of correlative microscopy methods. Together these studies will provide new information regarding the pleiomorphic structure of native RSV virions and the structural and functional role of the matrix protein in myxovirus assembly and morphology regulation. The two areas to be investigated in this project are: 1. Determine the native structure of RSV strain A2. Experiments will determine the natural structure of RSV through cryo-ET of frozen hydrated virions. Cryo-immuno-EM approaches and RSV RNA-specific probes will be used to define the placement and organizational patterns of the RSV structural proteins, F, G, SH, M, and the RNP complex during cryo-ET analysis of cryo-preserved virions. 2. Determine the structure of oligomeric RSV M in vivo and under in vitro assembly conditions. Experiments in this aim will determine the in vivo structure of RSV M in native virions and virus like particles (VLPs) through cryo-ET and sub-tomogram averaging. Cryo-ET, cryo-EM and helical reconstruction approaches will define the high-resolution structure of RSV M under in vitro assembly conditions.
This project addresses several questions of critical importance to the success of viruses that are pathogens of humans and other animals. Many viruses use a matrix or matrix-like protein to regulate assembly, budding, and overall virus particle structure. The structural studies proposed will first enable us to determine the native structure of RSV and the factors that govern RSV structure. The second set of studies has been developed to determine the structure of the oligomeric state of RSV M. RSV, the specific virus being analyzed is a human pathogen that causes bronchiolitis, viral pneumonia, and death in young children, the elderly, and immuno- compromised individuals. There is no vaccine against RSV and only one expensive prophylactic treatment available. The knowledge gained in the proposed study may be translated into the development of therapeutics and vaccines to improve public health.
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