Structural analysis of capsid and capsid-like proteins involved in viral morphogenesis using electron microscopy

Abstract

Viruses require specialised structural proteins for successful assembly and subsequent invasion of the host. These proteins may build the outer shells of viruses, encapsulate the genome, and interact specifically with host proteins. Detailed analysis of the structure and assembly of such proteins can provide valuable insights into viral morphogenesis, and may also direct us to possible therapeutic strategies against viral infection. This thesis is focused on characterising the structure and assembly of two proteins that are known to be critical for proper viral morphogenesis; the capsid-like immature poxvirus scaffolding proteins orfv075 and D13, and Rous sarcoma virus (RSV) capsid protein (CA). Poxviruses undergo complex and multi-stage morphogenesis. The scaffolding proteins dictate the size and shape of the immature poxvirus particles, and play a critical role in viral assembly through poorly understood mechanisms, hi this work, the assembly of the poxvirus scaffolding proteins orfv075 of orf virus and D13 of vaccinia virus were studied in vitro. These proteins assembled into spherical particles that resemble the authentic virion scaffold when introduced to Iow molarity buffers. The intrinsic curvature-inducing property of these proteins seems to facilitate membrane remodelling in the assembling immature virion, and the N-terminal domain appears to play a critical role in proper assembly. A 3D reconstruction of a D13 trimer was generated at ~20 A resolution using electron crystallography. The reconstruction showed structural similarity to a previously reported 3D model of an orfv075 trimer. Structural homology of these proteins to capsid proteins of nucleocytoplasmic large dsDNA viruses 0SΓCLDV) was apparent. Additionally, the organisation of the D13 and orfv075 trimers within 2D crystals models parts of an icosahedral capsid shell of a NCLDV. These results support the hypothesis that poxviruses share a common evolutionary lineage with large icosahedral viruses, and that the immature poxvirus scaffolding is the remaining trait of an ancient poxvirus precursor that may have adopted an icosahedral morphology. In a mature and infectious retroviral particle, CA forms a shell surrounding the genomic RNA and the replicative machinery of the virus. The irregular nature of this capsid shell precludes direct atomic-resolution structural analysis. This study developed an efficient in vitro protocol to examine the assembly of RSV CA, involving mild acidification. The resulting assembly products included planar sheets composed of CA hexamers; regular spherical particles with T=I icosahedral symmetry, built from CA pentamers; and particles resembling the authentic core of RSV, which are inferred to contain both pentamers and hexamers. The CA hexamers, which form the basal lattice of the capsid shell, exhibit strong similarities to hexamers found in other retroviruses, although full 3D reconstruction was hindered by a packing disorder within the sheets. The T=I icosahedral particles were subject to cryo-electron microscopy (cryo-EM) and image processing, and a pseudo-atomic model of the icosahedron was created by docking atomic structures of the constituent CA domains into the cryo-EM derived 3D density map at ~18A. The N-terminal domain (NTD) of CA forms pentameric turrets, which decorate the surface of the icosahedron, while the C-terminal domain (CTD) of CA is positioned undemeath, linking the pentamers. Biophysical analysis of the icosahedral particle preparation reveals that CA monomers and icosahedra exist in reversible equilibrium at pH 5, and suggests that RSV CA assembly is a nucleation limited process initiated by proton-linked CA dimerisation. Finally, an alternate in vitro CA assembly was developed, that depends on elevated temperature and salt concentration. With further exploration, this protocol is expected to add significantly to our understanding of retroviral capsid assembly.