Studies of virus assembly and maturation with four different systems are described in this competitive renewal proposal. P22 is a T=7 bacterial virus with nine gene products in the mature particle. Employing automated electron cryo microscopy and image reconstruction (cryoEM) an asymmetric structure was determined that allowed assignment of density to all the gene products and packaged dsDNA. We will continue our in vitro cryoEM work with available deletion mutants and assembly intermediates in order to understand scaffold protein-aided particle assembly, signal transduction of internal DNA pressures and particle reorganization following the loss of DNA. The studies will be extended in vivo with whole cell analysis of viral infection with high pressure freezing and tomography. We will visualize virus attachment, DNA delivery and virus assembly through time-course studies of infection. Studies of phage lambda will focus on correlating the structure of packaged DNA with measured internal pressures by employing available DNA packaging mutants that contain 90%, 80% 70% and 50% of the normal genome. Two archael viruses infecting the hyper thermophile sulfolobus (90?C and pH 2) are under study by cryoEM and crystallography. Sulfolobus Turistus Icosahedral Virus (STIV) is a T=31, 1000A, dsDNA virus containing 7 gene products, an internal membrane and an obvious relation to human adenovirus. We will build on our previous cryoEM and crystallographic studies of the particle and capsid protein by performing in vitro analysis of particle degradation with cesium sulfate and doing crystallography of ectopically expressed particle-associated proteins. Combining cryoEM and x-ray results will allow construction of a pseudo-atomic model of this bizarre virus. The availability of an infectious clone will allow the model to be tested. In vivo studies of virus infection and assembly will be studied with high pressure freezing of sulfolobus infected cells. Sulfolobus Islandicus Rodshaped Virus (SIRV) is a dsDNA virus. We will study in vitro assembly with dsDNA employing the available ectopically expressed capsid protein. The goal is to develop robust """"""""ropes"""""""" for nano technology applications and to understand the mechanism of assembly of a dsDNA virus rods. Finally we will study the ssRNA, T=4, tetraviruses. Penetrating new assays of particle maturation have allowed rapid screening of site-directed mutations that are followed by high resolution cryoEM, allowing a detailed understanding of electrostatically controlled maturation. ? ? ?
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