Structural characterization of nucleoprotein cores of human adenoviruses

Human adenoviruses (HAdVs) are large (MW ~150 mDa), complex, non-enveloped dsDNA viruses that cause acute respiratory disease, epidemic keratoconjunctivitis and gastrointestinal infections. Significantly, adenoviruses (AdVs) are being used as viral vectors for gene, vaccine and Oncolytic viral therapies. Although the in situ structures and arrangement of major and minor capsid proteins (CPs) in the assembled (icosahedral) viral capsid have been determined at near-atomic resolution, there is no information currently available on how the nucleoprotein core, comprising linear dsDNA (~36 kbp) and ?histone like? core proteins, is organized. The core density shows up as smeared ball in the icosahedral reconstructions of the AdVs. This is in contrast to characteristic spooled organization of the dsDNA seen in bacteriophages and herpesviruses. The details of nuclear core organization provide clues into the mechanism of genome packaging into AdVs, which is still unresolved. Here, we propose to employ emerging cryo-electron microscopy (Cryo-EM) methods to elucidate the structural organization of the AdV nuclear core by subtracting the icosahedral capsid regions from the particle (virion) images and reconstructing the core region(s) independently without imposing icosahedral symmetry. In Aim 1, we will reconstruct the nuclear core organization of mature HAdV5. Additionally, we will elucidate the reorganization of the nuclear core upon heat treatment that is known to mimic the structural changes that occur during partial disassembly and endosome escape. In Aim2 we will image the distinct nuclear core organization observed in hyper-thermostable and noninfectious form (ts1 mutant) of HAdV-C5 as well as elucidate the molecular basis of its greater thermo-stability of ts1 particles. Collectively, these studies provide structural details of nucleoprotein core organization in AdVs with implications for revealing the mechanism of genome packaging into AdVs as well as the changes that occur upon HAdV maturation. In addition, these studies will elucidate the changes that occur during the partial disassembly and infection of HAdV virions, emulated by the heat treatment.