Complex viruses such as HIV, herpesvirus and double strand (ds) DNA, tailed bacteriophages assemble through one or more intermediate stages before maturing to an infectious particle. The initial assembly complex recruits and organizes an inventory of components that spontaneously remodels its structure to define the energy landscape of maturation. Transitions between intermediates (called procapsids) have been characterized through changes in morphology, but there is virtually no mechanistic understanding about maturation pathways. In this proposal we seek to define a detailed structural/chemical mechanism for the maturation of the lambda-like dsDNA bacteriophage, HK97. Experiments will build on an atomic resolution model of the mature particle analogue, Head II (HII), prepared in an expression/assembly system that included only the capsid protein and virally encoded protease genes, and a 5.2A resolution crystal structure of a metastable intermediate designated Prohead II (PHII). Comparing the electron densities of the two particles (PHII 550A, HII 660A) revealed that the subunits remained largely rigid and underwent large translations and rotations with evidence for only local refolding.
Our aims for the next period of support are;(1,2) the high resolution crystal structure PHII and an early intermediate, Prohead I, that contains a putative scaffold-like polypeptide and viral protease, both of which are absent (due to hydrolysis by the protease) in PH II;(3) the high resolution crystal structure of the viral protease;(4) the high resolution structure of a late intermediate called EI-IV that is arrested in this state at pH 4;(5) single particle fluorescence studies of the Prohead II to the El-l transient intermediate to establish the nature of the cooperativity in the transition;(6) investigate DNA packaging with cryoEM structures of the P22 and HK97 connector complex and (7) study the dynamic behavior of the HK97 dynamic process with NMR and H/D exchange and mass spectrometry.
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