Location of the dsRNA-Dependent Polymerase, VP1, in Rotavirus Particles

Abstract

Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense, mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner, double-layered particle (DLP) into the cyotosol. DLP structures determined by x-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a “fivefold hub” of more structured density projecting inward from the VP2 shell of the DLP along each of the 12 fivefold axes. Analysis of the x-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the x-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the fivefold axis. The template strand enters from within the particle, and the dsRNA product of the initial, replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.

Rotavirus mRNAS are released by transcript-specific channels in the double-layered viral capsid

“…Our CID studies of three rotavirus transcripts that differ in sequence, type of proteins encoded, and length (S2, 2,691 nt; S6: 1,356 nt; S11: 667 nt, representing long, intermediate length, and short segments, respectively) provide unambiguous evidence that rotavirus channels specialize in extruding specific transcripts from DLPs. Our results provide direct evidence for the channel specialization model, which is also supported by the fact that ssRNA TCs are thought to form before replication within the capsid and that RNA TCs are very tightly packed within the viral particles (9,20). Furthermore, our results, along with studies showing that RNA segments interact with unique TCs close to an exit channel (21,22) while tightly packed in a semicrystalline state (23), preclude the possibility that extrusion of a specific transcript occurs through different channels each time.…”

“…Close to these channels, 12 TCs interact with the viral genomic segments and attach to a hublike structure formed by VP2 (5,6). This organization implies that 1 of the 12 TCs could be unoccupied (7)(8)(9).…”

Three-Dimensional Structure of a Protozoal Double-Stranded RNA Virus That Infects the Enteric Pathogen Giardia lamblia

“…4B). These regions might also be involved in interacting with and positioning the RdRp domain of the CP/RdRp subunit(s), as has been suggested, for example, for a similar N-terminal domain in rotaviruses (61,62).…”

Regulation of rotavirus polymerase activity by inner capsid proteins

“…Since the rotavirus genome consists of 11 segments of dsRNA, but 12 decamers are present in the core, one decamer may lack a VP1-VP3 complex. Estrozi and colleagues revealed the position and orientation of VP1 in the core [13••]. Their analysis indicates that VP1 is anchored to the VP2 decamer at a position slightly offset from the exact five-fold axis, barely covering the Type I channel.…”