Rotavirus non-structural proteins: structure and function

Abstract

The replication of rotavirus is a complex process that is orchestrated by an exquisite interplay between the rotavirus non-structural and structural proteins. Subsequent to particle entry and genome transcription, the non-structural proteins coordinate and regulate viral mRNA translation and the formation of electron-dense viroplasms that serve as exclusive compartments for genome replication, genome encapsidation and capsid assembly. In addition, non-structural proteins are involved in antagonizing the antiviral host response and in subverting important cellular processes to enable successful virus replication. Although far from complete, new structural studies, together with functional studies, provide substantial insight into how the non-structural proteins coordinate rotavirus replication. This brief review highlights our current knowledge of the structure-function relationships of the rotavirus non-structural proteins, as well as fascinating questions that remain to be understood.

Figure 1. The rotavirus replication cycle

The rotavirus triple layered particles (TLPs) first attach to sialo-glycans [78] or histo-blood group antigens (HBGAs) [79] on the host cell surface, followed by interactions with other cellular receptors, including integrins and Hsc70 [80]. Virus is then internalized by receptor-mediated endocytosis. Removal of the outer layer, triggered by the low calcium of the endosome, results in the release of transcriptionally active double-layered particles (DLPs) into the cytoplasm. The DLPs start rounds of mRNA transcription and these mRNAs are used to translate viral proteins. Once enough viral proteins are made, the RNA genome is replicated and packaged into newly made DLPs in specialized structures called viroplasms, which require lipid droplet components for formation [44]. The newly made DLPs bind to NSP4, which serves as an endoplasmic reticulum (ER) receptor, and bud into the ER. During this process, transient enveloped particles are seen in the ER. Removal of the transient membrane and assembly of the outer capsid proteins VP4 and VP7 result in the maturation of the TLPs. The progeny virions are released through cell lysis. In polarized epithelial cells, particles are released by a non-classical vesicular transport mechanism. IFN: interferon; PM: plasma membrane.

Figure 2. NSP3 and NSP2 structures

(A) The homodimer of the N-terminal RNA binding domain of NSP3 (residues 4–164) is shown in ribbon diagram in blue and golden yellow with a transparent surface. The 3' consensus sequence of rotavirus mRNA (5'-GUGACC-3') bound by NSP3 is shown in a yellow ball-and-stick representation, with oxygen atoms shown in red, and is indicated by an arrow (PDB ID: 1KNZ). (B) The structure of the NSP3 C-terminal domain (residues 206–315) homodimer in complex with eIF4G peptides is also presented in ribbon diagram with transparent surface. The NSP3 C-terminal domain homodimer is shown in blue and golden yellow, and the bound eIF4G peptides (in red and green) are indicated with arrows (PDB ID: 1LJ2). Structures of NSP2 (C–E) and its complexes with NSP5 (F) and RNA (G). The NSP2 monomer is shown in ribbon diagram, with its N- and C-terminal domains colored in purple and brown, respectively (PDB ID: 1L9V) (C). The catalytic cleft between two domains is indicated by an arrow. NSP2 self-assembles into octamers, as shown. The N-and C-terminal domains of one NSP2 molecule are colored and the remaining subunits in the functional octamer are shown in gray. The NSP2 octamer is shown as viewed along the 4-fold axis (D) and along one of the two 2-fold axes (E). Cryo-EM reconstructions of NSP2 with bound NSP5 (F, with NSP5 in blue) or RNA (G, with RNA in green) are shown as transparent surfaces with NSP2 (red ribbon) fitted into the density map.

Figure 3. NSP4

(A) Schematic of NSP4 functional domains and cellular and viral protein interaction domains. Oligomeric domains are mainly in the alpha amphipathic helix (H3) and the coiled-coil region. (B) Ribbon representation of the tetramer of SA11 NSP4_95–146 with a transparent surface (PDB ID: 2O1K). Two NSP4 molecules in an asymmetric unit are shown in blue and green, respectively. The red dot indicates the location of a divalent metal binding site, possibly binding Ca²⁺. (C) The pentameric structure of ST3 NSP4_95–146 is shown as a ribbon diagram with a transparent surface (PDB ID: 3MIW). Each NSP4 chain is colored differently [**77].