Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer

Abstract

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.

Understanding the penetrance of intrinsic protein disorder in rotavirus proteome

“…T = 1 symmetry of VP2 is needed for the initiation of the process of dsRNA synthesis [63]. Although the function of the N-terminal region (residues 1-132) is not understood well, this region seems to be important for the activation/ localization of the RNA-dependent RNA polymerase (VP1) and guanylyl transferase-methylase (VP3) into the virion center [64]. Our intrinsic disorder predisposition analysis revealed that the mean PPID of VP2 is 14.2% {Table 1}, indicating that it can be classified as moderately disordered protein.…”

“…RNA binding activity and incorporation of VP1 and VP3 into viruslike particles (VLPs) [64]. The principal domain of VP2 (residues~100-880) associated with the RdRp polymerase activity also exhibits several IDPRs (residues 100-120, 140-144, and 192-200) {Fig.…”

Mechanism of genome transcription in segmented dsRNA viruses

“…4.1 (see also color insert) (Prasad et al, 1988;Yeager et al, 1990;Prasad et al, 1996). The innermost capsid layer is formed by 120 molecules of a 102 kDa protein VP2 arranged as 60 dimers on a T=I icosahedral lattice (Lawton et al, 1997b). Such an organization with two molecules in the icosahedral asymmetric unit has also been referred to as a 'T=2' structure (Grimes et al, 1998).…”

“…In rotavirus, the inner capsid protein VP2 is proposed to have a dual role in defining the overall architecture of the virion, not only defining the icosahedral architecture of the capsid layers, but also acting as a scaffold for the proper assembly of the protein and nucleic acid compo-nents within the viral core (Prasad et alo, 1996;Lawton et al, 1997b). The inner capsid layer is formed from 60 dimers of quasiequivalent VP2 molecules organized on a T=I icosahedral lattice ( Fig.…”

“…In this illustration, the capsid region surrounding the icosahedral vertex has been computationally isolated from the three-dimensional structure of the TCP to reveal the locations of three distinct classes of VP2-dsRNA contacts (numbered as 1, 2, and 3) and the anchor point for the VP1/3 enzyme complex (arrow). Illustrations in (a), (b), and (e) adapted from Lawton et al (1997b). Used by permission.…”