Intracellular events in rotavirus replication

Abstract

l. A key step in the maturation of rotavirus is the intracellular budding event by which the mature virion is assembled. Immature inner capsid particles (ICPs) assemble in the cytoplasm of the infected cell adjacent to the rough endoplasmic reticulum membrane. The ICPs are then transferred from the cytoplasm to the lumen of the RER. This budding step is initiated by the interaction of the ICP with the cytoplasmic domain of the non-structural receptor glycoprotein, NSP4, which is inserted as a tetramer in the ER membrane (Au et al., 1989; Meyer et al., 1989). The overall aim of the work presented in this thesis has been to characterise the NSP4:ICP interaction. 2. A mutational analysis of the cytoplasmic domain of NSP4 was undertaken to identify regions of the protein that are essential for receptor activity. The requirement for the cysteine residues located in the third hydrophobic region of the protein, as well as the role of the C-terminal methionine residue, was assessed by dufung- site-directed mutagenesis. Transient expression of the mutant cDNA using recombinant T7 vaccinia virus was then used to assess the functional ability of the modified proteins. Substitution of serine for the cysteine residues did not affect receptor activity, but retention of the C-terminal methionine residue proved to be essential. Other mutants were constructed using PCR-based techniques to produce a range of NSP4 variants which were progressively truncated from the membrane-proximal (N-terminal) end of the molecule. These mutants were expressed as fusions with glutathione-S-transferase (GST) and a solid-phase binding assay was then developed to assess their ICP-binding activity. The results obtained indicate that the binding domain is relatively short and consists of most, if not all, of the C-terminal 20 amino acids including the final carboxy-terminal methionine residue. A mutant fusion protein presenting only ten of the C-terminal amino acids was receptor-negative. 3. The nature of the binding of ICPs to GST/NSP4 fusions was analysed by Scatchard analysis using receptor protein immobilised on the surface of glutathione agarose beads. All variant proteins exhibited comparable levels of binding activity providing that the C-terminal methionine residue was retained. The interaction was cooperative with an estimated Kd of the order of 7 x l0-11M, which agreed closely with the value of 5 x l0-11M measured previously for the membrane-anchored, full-length form of the protein. 4. Structural requirements for receptor activity were investigated by comparing the ICP-binding activity of NSP4 fusions where the carrier moiety was monomeric (maltose binding protein; MBP) or dimeric (GST). Receptor activity was almost totally abolished in MBP constructs leading to the inference that retention of biological activity is dependent on the formation of a multimeric assembly of NSP4. Limited proteolysis followed by mass spectrometry was used to identify the extent of the unstructured region at the carboxy-terminus. The results indicate that the carboxy-terminal region of the receptor contains a protease-sensitive domain composed of the C-terminal 29 residues. The 20 amino acid ICP-binding domain identified by mutational analysis is contained within this region. 5. An assay utilising radioactively labelled receptor protein was developed to characterize NSP4:ICP complexes. No evidence for a stable, specific interaction was obtained when soluble forms of the receptor were used, suggesting that soluble receptor possesses a lower affinity for the ICP than is the case for membrane or bead-immobilised forms. However, a specific, saturable association between the receptor and the ICP was achieved when the GST/C9O form of the receptor was bound to glutathione agarose beads prior to interaction with the ICPs. Analysis of these receptor:particle complexes indicated that they contained approximately forty C90 tetramers per ICP. 6. Preliminary work was initiated to characterise the structural details of the receptor footprint on the ICP. Examination of receptor:ICP complexes by transmission electron microscopy of negatively stained samples, or cryoelectron microscopy of frozen-hydrated specimens, revealed that differences in the appearance of decorated and native ICPs were subtle. Three-dimensional image processing of cryoelectron micrographs did not demonstrate the presence of any additional density attributable to bound receptor, despite biochemical confirmation of the presence of receptor in the same decorated samples. 7. The results obtained in this study are discussed in terms of a model for the structure of the cytoplasmic domain and the implications of the NSP4:ICP interaction for the membrane budding process by which rotavirus matures.