Abstract:
Rotaviruses are non-enveloped icosahedral viruses that belong to the family Reoviridae. The viral RNA polymerase resides within a transcriptionally active, subviral particle, termed the double-layered particle (DLP). Rotavirus DLPs are composed of two protein capsid layers; an outer layer of 260 VP6 trimers, and an inner core of VP2. The VP2 core encapsidates the segmented dsRNA genome and the endogenous transcriptase machinery (VP1 and VP3). During infection, the DLP is released from the infectious triple-layered particle (TLP) within the cytoplasm and synthesises mRNA transcripts. Following assembly of progeny DLPs, these particles are targeted to the endoplasmic reticulum (ER) via an interaction with a virally encoded non-structural protein, NSP4. The interaction of the particle with the cytoplasmic domain of NSP4 precedes translocation of particles across the ER membrane into the lumen and assembly of the outer capsid layer.
This research project has investigated changes in the structural and catalytic properties of rotavirus DLPs induced by either a recombinant soluble form of the NSP4 cytoplasmic domain or subgroup-specific monoclonal antibodies that recognise and bind to discrete epitopes on VP6. NSP4 was found to cause a profound alteration in the biophysical properties of the particle, indicative of a conformational change in the capsid. Consistent with this interpretation, the transcriptase activity of the DLP was significantly reduced in the presence of NSP4. The changes to the particles were dependent on the presence of an oligomeric form of NSP4 and were not observed in the presence of soluble monomeric NSP4 peptides that retain the ability to bind the DLP.
RNA transcriptase activity was also significantly inhibited when DLPs were incubated with Fab fragments from subgroup (SG) I-specific monoclonal antibodies but not SG II. Cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction revealed that SG I-specific Fab fragments preferentially bind to the five VP6 trimers surrounding the type I channel (the site of mRNA release), potentially causing constriction of the type I channel. Three-dimensional reconstructions of Fab-decorated DLPs also demonstrated that the binding "footprints" of both the SG I and II specific Fab fragments are comprised of residues from two monomers within each VP6 trimer.
DLPs belonging to SG II were observed to possess a markedly reduced level of transcriptase activity when compared to SG I particles. Structural analysis revealed an absence of VP6 trimers at the five positions surrounding the type I channel as compared to SG I DLPs. The absence of these trimers may be the cause of the lowered rate of RNA production.
Taken together, the findings of this research demonstrate the structural plasticity of the rotavirus DLP and show that subtle conformational changes can influence the endogenous RNA transcriptase activity of the particle. The binding of α-SG I Fab fragments to UK bovine DLPs produces similar inhibition of transcriptase activity, suggesting comparable mechanisms may function in either case.