Date Published: April 20, 2017
Publisher: Public Library of Science
Author(s): Courtney P. Long, Sarah M. McDonald, Katherine R. Spindler.
Viruses are obligate intracellular parasites comprised of a nucleic acid genome (RNA or DNA) that is encased within a proteinaceous capsid particle and/or lipid envelope. Genome replication and virion assembly are central processes in the life cycles of all viruses. In many cases, a virus will first make multiple copies of its genome and then subsequently package those copies into newly formed viral capsids or envelopes. However, rotaviruses and other members of the Reoviridae family differ in that they replicate their genomes in concert with virion assembly. Specifically, the segmented, double-stranded RNA (dsRNA) rotavirus genome is copied within a subviral assembly intermediate that goes on to become a mature, infectious virion. A key feature of this replicase–assembly process is that the rotavirus polymerase (VP1) is only active when tethered to the core shell protein (VP2) within the confines of an assembly intermediate. Yet several gaps in knowledge exist about the structure and composition of early assembly intermediates for rotavirus, and the mechanism by which VP2 engages and activates VP1 is not completely understood.
The rotavirus virion is a triple-layered particle (TLP) ~85 nm in diameter, and it is made up of a VP2 core shell, a middle VP6 layer, and an outer VP7 layer that is embedded with VP4 spike attachment proteins (Fig 1A) [1–2]. It is thought that the VP1 polymerase forms a complex with the VP3 RNA capping enzyme and that VP1–VP3 heterodimers are tethered to VP2 beneath each of the 12 icosahedral fivefold axes . The rotavirus genome is made up of 11 dsRNA segments, coding for 6 structural proteins (VP1–VP4, VP6, and VP7) and 6 nonstructural proteins (NSP1–NSP6) . The TLP is the infectious form of the virus that attaches to and enters into host cells. However, during the cell entry process, the outer VP4–VP7 layer of the TLP is shed, depositing a double-layered particle (DLP) into the cell cytoplasm. VP1 polymerases within the DLP synthesize single-stranded, positive-sense RNAs (+RNAs), which acquire a 5’ cap structure (m7GpppG) by the activities of VP3 . These +RNAs serve as mRNA templates for protein synthesis, and they are also selectively assorted and packaged into an early assembly intermediate where they serve as templates for genome replication by VP1 (see details below) . The mechanism by which rotavirus acquires one of each of its 11 genome segments is poorly understood, yet studies of other Reoviridae members suggest that this assortment process is mediated by RNA–RNA interactions among the single-stranded transcripts [6–7].
Atomic resolution structures have been determined for rotavirus TLPs and DLPs, revealing exquisite details about capsid protein organization [1–2, 8]. By contrast, much less is known about the structures of early, replicase-competent assembly intermediates for rotavirus. One reason for our lack of knowledge about these particles is the fact that they are encased within viroplasms, which are discrete, cytoplasmic inclusions ~1–3 μm in diameter (Fig 1B) . At <100 nm in diameter, assembly intermediates are too small to be seen using conventional light microscopy, and unfortunately, viroplasms are so electron-dense that internal features can’t be resolved by higher-resolution electron microscopic (EM) imaging (Fig 1C). Subviral particles capable of mediating in vitro dsRNA synthesis can be isolated from rotavirus-infected cells using biochemical approaches [10–13]. These putative early assembly intermediates contain VP1, VP2, VP3, and VP6, as well as NSP2, a multifunctional viral nonstructural protein critical for viroplasm formation and genome replication . When viewed using negative-stain EM, the isolated particles are heterogeneous in their sizes and features (Fig 1D) . Specifically, the smaller particles (~30–40 nm in diameter) exhibit smooth borders, whereas the larger particles (~50–70 nm in diameter) show a rough, honeycomb pattern on their surface, reminiscent of 80-nm DLPs. Unlike DLPs, however, the isolated assembly intermediates are very fragile and highly permeable to metal stains and RNases, suggesting that they do not have fully formed capsid layers. One hypothetical model of the rotavirus replicase–assembly pathway is that the smaller, smooth particles turn into the larger, rough particles and ultimately into DLPs (Fig 1D). For instance, the ~30-nm smooth particle could represent the earliest rotavirus assembly intermediate, within which genome replication is initiated by VP2-bound VP1. Biochemical data suggest that each VP1 polymerase functions independently within an assembly intermediate but that 11 polymerases act in synchrony with each other so that the genome segments are synthesized at the same time . It is not known how the activity of one polymerase is coordinated with those of the other polymerases. Nevertheless, as the polymerases convert the +RNAs into dsRNAs, the particle presumably expands and begins to acquire a VP6 layer, forming a larger rough particle. Prior to final DLP assembly, the nonstructural protein NSP2 must be removed. Nascent DLPs egress from the viroplasm and bud into the adjacent endoplasmic reticulum, where they are converted into TLPs by addition of VP4 and VP7 [15–16]. Further studies are required to elucidate higher resolution structures, compositions, and activities of isolated rotavirus assembly intermediates and to test this hypothetical model of early morphogenesis. To initiate genome replication (i.e., dsRNA synthesis), the VP1 polymerase must be bound by the core shell protein VP2. In DLPs or TLPs, VP2 is organized as 12 interconnected, decameric units (Fig 2A), but its structural organization in assembly intermediates is not known [1–2,8]. The VP2 monomers in each decamer unit adopt one of two slightly different conformations (VP2-A and VP2-B). One conformation, VP2-A, converges tightly around the icosahedral axis, whereas the other conformation, VP2-B, intercalates between adjacent VP2-A monomers. The extreme N-terminal region of VP2 (residues ~1–100) protrudes inward and makes contact with VP1 (Fig 2B). Estrozi et al. predicted that VP1 is positioned against the inner surface of the VP2 core shell off-center from the fivefold axis and that it is stabilized against the core shell by VP2 N termini . The regions of VP2 that contact VP1 in the DLP structure are the same as those shown to be important for VP2-mediated VP1 enzymatic activation in vitro . This observation suggests that the VP1 and VP2 binding interaction during the early replicase–assembly process may be similar to the VP1 and VP2 binding interaction during transcription. During their intracellular life cycles, viruses make numerous copies of their nucleic acid genomes and package them into nascent particles. Viral genome replication and particle assembly are often highly coordinated within the infected cell to maximize efficiency. Rotaviruses and other Reoviridae family members may very well exhibit the utmost level of coordination, as they replicate their genomes concurrent with assembly of new virions. The mechanism of this concerted replicase–assembly process is not completely understood. Isolated rotavirus subviral particles that can perform dsRNA synthesis in vitro are just beginning to be characterized in terms of their structure and composition, and there is much to be learned about how the activity of the rotavirus VP1 polymerase is regulated via interaction(s) with the core shell protein VP2 in the context of the assembling particle. Although other viruses do not perform this same multitasking feature of replicating their genomes while assembling particles, it is apparent that they also must regulate the activities of their polymerases. The vast majority of viral polymerases do not function as sole polypeptides during infection . Instead, they are components of multisubunit complexes, and interactions between the protein constituents dictate the polymerase activity. Thus, studies of rotavirus polymerase regulation during particle assembly may broadly inform an understanding of how other viruses ensure that genome replication occurs at the right place and time in the infected cell. Source: http://doi.org/10.1371/journal.ppat.1006242