Date of Award
Level of Access
Doctor of Philosophy (PhD)
Biochemistry and Molecular Biology
S. Monroe Duboise
Second Committee Member
Third Committee Member
A fundamental step in the replication of a viral particle is the self-assembly of its firm capsid shell from the constituent proteins, usually consisting of one or a few protein types. For most viruses, the capsid possesses an icosahedral lattice which varies in size and complexity to accommodate the genomic material. At large, the process that results in the formation of the nucleocapsid is unique to each virus family. Nonetheless, recent structural and genetic studies indicate a common capsid-assembly is shared by dsDNA tailed bacteriophages of prokaryotes and animal viruses of the family Herpesviridae and Adenoviridae. These viruses assemble an empty immature capsid shell by successive addition of structural capsid proteins around a scaffolding protein core, which is then expelled from the interior to accommodate the viral genome, packaged through a small portal channel. Examined in this publication are the structural components of the capsid and the morphogenesis process of two viruses, the Murid Herpesvirus 4, strain 68 (MHV- 68) and haloalkaliphilic bacteriophage ϕlN2-2, a siphovirus isolated in our laboratory from the Kenyan soda-lakes. ϕ1N2-2 infects native Idiomarina-like bacteria species.
Components of the MHV-68 capsid are identified by genetic means and tested for their properties to aggregate into discrete puntate intracellular distribution, characteristic of virus liker particles or assembly factories. In the case of the most divergent capsid component, here we demonstrate by indirect immune-detection methods that ORF65 protein is located on the surface of the capsid, bound to the major capsid protein, and thus it represents the functional homolog of the herpesvirus small capsid protein. We further propose that, unlike its herpesvirus counterparts, this small capsid component may be required for capsid assembly. Using a functional recombinant fluorescently tagged- ORF65 protein, we studied the controlled incorporation of this protein to nascent capsid at the nucleus, and determined that the N-terminal half of the protein is sufficient to bind to the nascent capsid. Ultrastructural examination, using transmission electron microscopy, of infected cells and TPA-reactivated MHV-68 latently infected Sll cells, in combination with immune-detection of capsids with an anti-ORF65 antibody, indicates that large (2 μm) proteinaceous aggregates are formed during viral replication. These aggregates are presumably capsid assembly factories. Moreover, we demonstrate that TPA-reactivation from Sll, resulted in cellular changes associated with loss of nuclear membrane integrity and the premature presence of immature particles in the cytosol. We interpret this abnormality as either the effect of rapid viral reactivation, in contrast to lytic infection, or to the direct effect of TPA.
The genomic analysis and genetic basis of virus replication of soda-lake phage ϕlN2-2 is described. The genome is functionally organized: Head capsid and tail morphogenesis, DNA replication and regulation, and a series of novel small genes, which share significant sequence only to a second soda-lake phage ϕlM2-2 of Idiomarina-like bacterial hosts. Comparative genetic analysis of the capsid genes of ϕlN2-2 and other soda-lakes phages suggest that they share common structural features, besides the lack of sequence similarities. Given that these bacterial viruses are adapted and able to tolerate the extreme alkaline hyper saline environment of the soda lakes, we analyze the structure of the capsid, as the viral compartment which protects and transport the infectious genome. Capsid protein of soda-lake viruses contain a higher number of acidic residues and are predicted to have more negatively charge surfaces, when compared to the closest non-alkaliphilic virus counterpart in GenBank. Presumably, this suggests that a strategy used by viruses to adapt to the extreme alkaline environment of the soda-lake is to accumulate acidic residues to confer a negative-electrical charge of the surface of the capsid.
In Chapter 5, we describe a conserved process of capsid-morphogenesis utilized by the alkaline phage ϕlN2-2. The basic capsid components, portal-protease-MHP (major head protein), share significant sequence similarities and are structurally homologous to the capsid subunits of the prototypical coliphage HK97. In the absence of other viral component, recombinantly expressed ORF10 gene product (gp10) is sufficient and required to self-assemble into a T=7 (420 subunits) symmetrical capsid precursor. Similar to phage HK97gp5 capsid protein, the N-terminal domain of gp10 is transiently required to guide capsid assembly, but is proteolytically removed by an incorporated gp9- capsid maturational protease upon completion of the capsid shell in vitro, and presumably concomitant with DNA packaging in vivo. To guide capsid assembly, scaffolding domains of different gp10 subunits interact through an endogenous α-helical coiled-coil motif. This self-interaction property of the scaffolding protein, and particularly that of the coiled-coil motif, is exploited as a molecular signal to package foreign protein in the inner cavity of the self-assembled capsid-like particles, providing a platform to potentially utilize the recombinant capsid of ϕlN2-2, and related viruses, as a nanoparticle-based delivery system, where the incorporated molecule of interest is protected by the protein shell. Alternatively, we demonstrate that the C-terminus of the 415 copies of gp10 is present on the surface of assembled capsid; and thus it can be genetically modified to display foreign polypeptides without interfering with the process of capsid self-assembly or maturation. Overall, recombinant ϕlN2-2 base capsids offer a novel nanomolecular platform for biomedical or biotechnology applications.
Lobo-Galo, Naun, "Structural and Morphogenesis Analysis of the Capsid of the Murid Herpesvirus 4, Strain 68 (MHV-68) and the Alkaliphilic Bacteriophage ϕ1N2-2" (2013). Electronic Theses and Dissertations. 1932.
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