Structural Biology Of Virus Assembly

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

We seek to identify features with potential for antiviral drug and vaccine interventions, and to elucidate mechanisms at play in the assembly, maturation, and activation of large macromolecular complexes in general. We have a major interest in hepatitis B virus, a major human pathogen, and in retroviruses, primarily HIV-1. The latter (HIV-related studies) is the subject of a separate report (AR041116-27). Hepatitis B virus has infected approximately one third of the human population and causes almost 1 million deaths from liver disease annually. The capsid is a defining feature of a virus, distinct from host components, and therefore a target for intervention. Unusually for a virus, Hepatitis B assembles two capsids, with different geometries, from the same dimeric protein. Geometric principles dictate that the subunits in this system occupy seven different environments. We compared the two capsids by cryo-electron microscopy under identical conditions and found that the polypeptide chains adopt seven different conformations. These structures were used to calculate potential energies (analogous to elastic deformation or strain) for the individual chains, dimers, and several higher-order groupings discernible in the two lattices. We also calculated the binding energies between chains. We found that some groupings have substantially lower energy and are therefore potentially more stable, allowing us to predict likely intermediates on the two assembly pathways. We also observed such intermediates by electron microscopy of in vitro capsid assembly reactions. This is the first structural characterization of the early assembly intermediates of this important human pathogen. Encapsulins are protein nanocompartments that are widespread in bacteria and archea. Recently, their potential applications in biomedical research and targeted drug delivery have gained much attention. The M. xanthus encapsulin system contains the encapsulin promoter EncA and three cargo proteins, namely EncB, EncC and EncD. Both EncB and EncC are like bacterial ferritins which are capable of oxidizing Fe+2 to less toxic Fe+3. This ferroxidase activity coupled with the high Fe+3 storage capability of the EncA shell protects the bacteria against oxidative stress. We used a combination of cryo-EM and x-ray crystallography techniques to analyze the structural features of the EncB and EncC cargo proteins and their binding to the EncA shell. Our results show that both EncB and EncC are decameric ferritin-like proteins with diiron binding sites. Comparison of EncB and EncC ferroxidase centers show carboxylate ligand shifts in Fe bound forms. We have also shown that both cargo proteins are located at the 5-fold-axes within the shell and in EncB the targeting peptide binding sites extend to the EncA promoters surrounding the 5-fold-axes. These structural studies should form a basis for design and engineering of encapsulin based systems for biomedical applications. A manuscript describing these studies was published.