Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Curr Opin Virol. 2012 May 16;2(4):373–379. doi: 10.1016/j.coviro.2012.04.005

Interactions among Capsid Proteins Orchestrate Rotavirus Particle Functions

Shane D Trask 1, Kristen M Ogden 1, John T Patton 1,*
PMCID: PMC3422376  NIHMSID: NIHMS373939  PMID: 22595300

Abstract

Rotaviruses are members of the Reoviridae family of non-enveloped viruses and important etiologic agents of acute gastroenteritis in infants and young children. In recent years, high-resolution structures of triple-layered rotavirus virions and the constituent proteins have provided valuable insights into functions. Of note, structural studies have revealed the position of the viral RNA-dependent RNA polymerase, VP1, within the inner capsid, which in turn provides clues about the location of the viral capping machinery and the route of viral transcript egress. Mechanisms by which the viral spike protein, VP4, mediates receptor binding and membrane penetration have also been aided by high-resolution structural studies. Future work may serve to fill the remaining gaps in understanding of rotavirus particle structure and function.

Introduction

Virus capsids protect the viral genome and mediate entry into target cells. The segmented, double-stranded (ds) RNA genomes of Reoviridae viruses are packaged into multi-layered, icosahedral capsids that never fully disassemble; instead, RNA transcripts that direct viral protein expression and genome replication are synthesized within the confines of a subviral particle and extruded into the cytosol. Recent technological advances have enabled determination of high-resolution structures for the virion of rotavirus, the medically most relevant of the Reoviridae viruses. In combination with biochemical studies, virion structures have clarified many aspects of rotavirus biology, including the interaction of the viral polymerase with the inner capsid protein during transcription and replication and the conformational changes in the outer capsid that accompany trypsin proteolysis (maturation) and membrane penetration during entry.

Overview of the rotavirus virion

The inner capsid of the rotavirus virion is formed by VP2 (102 kDa), a comma-shaped, plate-like protein (Fig. 1A–B) [1,2]. In two alternate conformations (A and B), VP2 forms a pseudo T=1 capsid from 60 asymmetric dimers: VP2-A converge around the icosahedral five-fold axes, while VP2-B interdigitate between the VP2-A and sit farther away from the five-fold axes (Fig. 1B) [1]. It is convenient to think of the VP2 capsid as twelve decameric arrangements of VP2, each centered on an icosahedral five-fold axis of rotation (Fig. 1B). Both the multimeric configuration and the monomeric fold of VP2 are highly conserved among the inner capsid proteins of the Reoviridae [1,3,4]. Packaged within the VP2 capsid are the eleven dsRNA segments (ranging from 0.7–3.3 kbp) of the rotavirus genome, roughly arranged as concentric spheres [5]. One copy each of the viral RNA-dependent RNA polymerase (RdRp), VP1 (125 kDa), and the RNA capping enzyme, VP3 (88 kDa), is thought to be associated with each genome segment. The VP1-VP3 pairs are anchored to the interior side of the VP2 capsid through contacts made near the five-fold axes. Because there are eleven genome segments, but twelve vertices within the VP2 capsid, one of the five-fold positions may be “empty”, lacking an associated VP1 and/or VP3 component.

Figure 1. Overview of the rotavirus virion.

Figure 1

Open in a new tab

(A) Hybrid view of the rotavirus virion (PDB IDs 3IYU, 3N09, and 2R7U) [9,13]. The outer capsid proteins of the triple-layered particle (TLP), VP4 (red) and VP7 (yellow), mediate attachment and entry of the virus into target cells. Entry releases a double-layered particle, whose outer surface is composed of VP6 (green), into the cytosol. The inner capsid protein, VP2 (blue), encloses the dsRNA genome and multiple copies of the viral polymerase, VP1 (pink), and capping enzyme, VP3 (not shown). (B) View of an icosahedral five-fold axis from inside the particle. Each five-fold axis is immediately surrounded by 5 copies of VP2 (VP2-A; light blue), while the alternate conformation of VP2 (VP2-B; dark blue) sit farther away and interdigitate between VP2-A. Current evidence indicates that VP1 (pink silhouette) can occupy one of five rotationally symmetric positions near the five-fold axis. Note the channel formed at the five-fold axis (obscured by VP1). The channel is thought to be the exit for [+]RNAs during transcription. (C) Enlarged side view of the capsid. Note that several trimers of VP6 contact each VP2 molecule. The pear-like shape of VP6 forms large depressions in the DLP surface; the pseudo-six-fold symmetric depressions that flank each five-fold axis allow docking of VP4 trimers deep into the VP6 capsid. Trimers of VP7 latch onto each VP6 trimer and partially obscure the depressions in the VP6 layer, thereby preventing VP4 dissociation.

Surrounding the VP2 capsid and its contents is a T=13 layer formed by 260 trimers of the VP6 protein (45 kDa; Fig. 1A) [2]. VP6 subunits intertwine in a right-handed twist, forming a roughly pear-shaped trimer [6]. VP6 shares structural similarity with the orthoreovirus (reovirus) and aquareovirus membrane penetration proteins [6–8], but instead functions as an adapter for the rotavirus outer capsid proteins (VP4 and VP7) that mediate entry. Trimeric spikes of VP4 (87 kDa) are anchored into depressions in the VP6 layer that surround the five-fold icosahedral axes (Fig. 1A, C) [9]. Up to 60 spikes can potentially decorate the virion, although the precise occupancy of VP4 may vary among rotavirus strains. VP4 has receptor binding activities and functions as the membrane penetration protein during entry. The trimeric glycoprotein VP7 (~ 34 kDa) covers the virion surface and locks the VP4 spikes into place (Fig. 1A, C) [9]. VP7 trimerization is entirely dependent on coordination of calcium ions that mediate the interactions between each subunit [10]. A VP7 trimer sits atop each VP6 trimer, arranged in the same T=13 configuration, completing the outer capsid [9,11]. The rotavirus triple-layered particle (TLP) is the infectious form of the virion and measures approximately 100 nm in diameter, spike-to-spike (Fig. 1A). Outer capsid proteins VP4 and VP7 are shed during entry, delivering a double-layered particle (DLP) to the cytosol to initiate infection (Fig. 1A).

The inner capsid orchestrates transcription

Transcription of rotavirus positive-sense RNAs ([+]RNAs) by VP1 occurs within the DLP [12]. Since little structural information about VP1 could be gleaned from virion reconstructions alone, structures of purified VP1 were solved [13]. VP1 is a roughly globular protein with a large central cavity that houses the active site for RNA polymerization. The VP1 polymerase domain has a “cupped right-hand” architecture common to polymerases, but large N- and C-terminal domains (common to the Reoviridae) form a “cage” that encloses the VP1 catalytic center [13]. Four tunnels, each with a discrete function, permit access to the active site (Fig. 2A): (1) entry of the template RNA, (2) exchange of nucleotides and pyrophosphate during catalysis, (3) egress of the template RNA (during transcription) or dsRNA (during genome replication), and (4) release of [+]RNA during transcription.

Figure 2. Interactions among the inner capsid proteins during [+]RNA transcription.

Figure 2

Open in a new tab

(A) Model for transcription of capped (red circle) rotavirus [+]RNAs (yellow), based on empirical evidence and modeling on the capsid and polymerase proteins of reovirus (PDB IDs 1EJ6 and 1UON) [4,14–16]. VP1 (pink) binds the VP2 (blue) capsid, simultaneously interacting with multiple VP2 molecules (see panel B). Structural information, and comparison with the RdRP of reovirus indicate the functions and orientations of the four tunnels of VP1 that direct substrates in and out of the central active site: 1. Entry of the template negative-sense RNA (“−”, blue); 2. Exchange of nucleotides and pyrophosphate; 3. Egress of the template RNA; 4. Release of [+]RNA. It is thought that a “cap-binding site” near tunnel 1 allows VP1 to retain the cognate [+]RNA to the template during transcription. The presumed location of VP3 is indicated. The proximity of VP1 tunnel 4 to the five-fold channel indicates that VP3 must quickly bind and cap (white arrow) the nascent [+]RNA before it is extruded out of the virion. (B) Detailed view of the VP1-VP2 interaction. The inset panel represents an approximately 90° vertical rotation of the view in (A). VP2-B conformers are shown in ribbon to highlight the N-terminal arms (yellow) that project towards the five-fold axis and VP1 (the arms of VP2-A are disordered and not observed in particle reconstructions). VP1 is shown in ribbon and colored according to the three primary domains described in the text: the Reoviridae-specific N-terminal (green) and C-terminal (magenta) domains that flank the central polymerase domain (pink). Rotation (~45°) and magnification of the inset view provides greater detail of the VP2-B N-terminal arm that approaches and likely interacts with VP1 (white arrowhead in inset).

At the 12 five-fold axes of icosahedral symmetry (each corresponding to the ‘center’ of a VP2 decamer) are small channels in the virion that permit extrusion of [+]RNAs (Fig. 1B). The diameter of these channels is larger in the DLP than in the TLP, suggesting that removal of VP7 permits conformational changes that trigger transcription [11,14]. It has been inferred that both VP1 and VP3 must occupy positions near the five-fold channels to direct nascent transcripts out of the DLP. Consistent with this model, the RdRp (λ3) of reovirus is located proximal to, but off-center-from, the five-fold channels of the virion [15]. Fitting of the VP1 crystal structure into rotavirus reconstructions has confirmed that VP1 adopts a similar orientation and association with the VP2 capsid (Fig. 1B, 2A) [16]. This position and orientation enable VP1 to direct transcripts towards the five-fold channel (Fig. 2A). Mutagenesis of VP2 mapped the site required for activation of dsRNA synthesis by VP1 to the “apical” and “central” domains of VP2 [17], precisely the region of VP1 binding identified in particle reconstructions, which suggests that VP1 interacts with the same site on the VP2 capsid during both transcription and genome replication. The striking similarity between the reovirus core-λ3 and rotavirus VP2-VP1 interactions suggest that the Reoviridae may have a highly conserved capsid-RdRP interaction mechanism that functions to promote virion assembly and to direct [+]RNAs during transcription. The N-termini of the VP2 proteins are not fully resolved in virion reconstructions, but the resolved regions are directed towards the five-fold axis of each decamer (Fig. 2B) [1]. It appears as though the VP2 N-termini directly contact the bound VP1 on one side, suggesting that these appendages may function to support or orient the RdRp (Fig. 2B; S. Harrison, personal communication). Accordingly, biochemical analyses suggest that the VP2 N-termini are required for VP1 encapsidation [18]. VP2 N-termini vary in sequence and length between rotavirus strains but generally contain numerous positively charged amino acids [19]. Thus, a primary function may be to interact with [+]RNAs during virion assembly, dsRNAs within the assembled core, or both.

The least well understood aspect of the rotavirus polymerase complex is the RNA capping enzyme, VP3. The structure of VP3 is not known, nor has it been located within virion reconstructions. However, multiple enzymatic RNA capping functions, including guanylylation, N7-methylation, and 2′-O-methylation, have been assigned to VP3 [20,21]. The structure of bluetongue virus (BTV) VP4, the functional homolog of VP3, has been solved [22]. Based on sequence alignments and structure predictions, VP3 is anticipated to adopt a structure similar to BTV VP4, particularly with regard to the predicted methyltransferase domains (K. Ogden, unpublished data). VP3 is larger than BTV VP4; yet, VP4 has been shown to encode an enzymatic activity (RNA 5′-triphosphatase) that has not been observed for VP3 in vitro [20,23,24]. The localization of VP1 near the five-fold channels of the virion necessitates that VP3 must occupy an adjacent position (Fig. 2A). The [+]RNA exit tunnel of VP1 is oriented near the five-fold channel (S. Harrison, personal communication). Unlike the capping enzyme turrets of reovirus [4,15], which sit on the exterior of the inner capsid, VP3 must intercept and modify the 5′ end of rotavirus [+]RNAs shortly after transcription is initiated. Consistent with this idea, biochemical data indicate that rotavirus [+]RNAs are capped as they reach only five to seven nucleotides in length [25]. Conformational flexibility or positional variability are likely reasons for the inability to detect VP3 in rotavirus particle reconstructions, which rely on icosahedral symmetry and averaging functions to observe proteins at near-atomic resolution. It is likely that VP3 has a transient interaction with VP1 and [+]RNAs, as it must quickly bind, cap, and release RNAs as they are directed out of the core and into the cytosol. A prolonged or highly stable interaction with RNA (or VP1) would presumably slow the rate at which rotavirus could generate and release [+]RNAs. These observations, as a whole, suggest several challenges that must be addressed to better understand VP3, an essential component of the rotavirus replication machinery.

Interactions among the outer capsid proteins prime the spike for entry

Outer capsid assembly appears to be a step-wise process in which VP4 is added to an assembled DLP, and subsequent budding through the endoplasmic reticulum membrane provides access to VP7, which locks VP4 in place [9,26,27]. The trimeric VP7 structure has been solved, both as a soluble protein bound by a neutralizing antibody and assembled onto the virus particle [9–11]. The VP7 trimer is a triangular plate with a slight depression in the center (Fig. 3Aii). Two calcium ions are bound between subunits near each of the corners of the triangle [10]. VP7 assembles onto the DLP using flexible N-terminal arms that latch onto VP6 [6,11]. VP7 functions as a molecular switch during entry. As trimers uncouple (likely due to calcium depletion within an endosomal vesicle) and dissociate from the virion surface, they cue membrane penetration by the mature form of the VP4 spike [28–30].

Figure 3. VP7 interacts with VP5* to regulate structural transitions of the spike.

Figure 3

Open in a new tab

(A) Three-quarters profile of the rotavirus capsid. A single VP4 spike trimer (red/brick/orange) is embedded in the VP6 (green) capsid. The A and B subunits of the spike project away from the virion, while the C subunit lies nearly flat to the particle surface. The distal tips of the spike are formed by two globular domains of VP8* (pink; inset i), which bind sialic acid (SA) moieties (or other glycans) to mediate attachment of the virion to target cells (PDB IDs 1KQR and 3IYU). The VP7 (yellow) layer binds VP6 and encloses the VP4 base to restrict its dissociation from the virion. Note the N-terminal arms of VP7 that extend down and latch onto VP6; this is the primary mode of VP7-VP6 interaction [11]. VP7 trimers are stabilized by two bound calcium (Ca2+) ions at each subunit interface (inset ii). (B) Top view of the VP7 capsid surrounding the VP4 spike, highlighting the asymmetry that influences the spike conformation. Relative to the view in (A), the structure has been tipped towards the viewer. The two VP7 trimers immediately counter-clockwise of the black-and-white arrowhead are not shown in (A). The arrowhead indicates a gap in between two adjacent VP7 trimers that imparts asymmetry on the spike binding site and permits interaction of VP5*C with the two VP7 trimers on either side of the gap (see black-and-white arrowhead in (A)) [9]. In this view, the outer features of the spike complex have been truncated to highlight both the trimeric configuration of the spike base, and to illustrate how VP7 occludes the base, preventing dissociation of the spike.

To become fully infectious, the virion must be released from the cell, and VP4 must be proteolytically cleaved by trypsin-like proteases in the intestinal lumen. Prior to trypsin cleavage, the VP4 spikes are flexible and cannot be observed in particle reconstructions. Proteolysis triggers a reorganization in which the spike adopts a distinct, rigid conformation [31]. VP4 is cleaved within a defined region to generate two fragments (VP8* and VP5*) that remain non-covalently associated. VP8* (~ 27 kDa) is the smaller fragment derived from the VP4 N-terminus. The structures of the globular domain of VP8* from several rotavirus strains have been solved; all have a galectin-like fold, and many have been shown to bind cellular glycans [32–34]. VP8* has been shown to bind terminal sialic acid residues in many rotavirus strains (Fig. 3Ai) [33]. Recently, though, it has been reported that certain strains, notably those that infect humans, bind a broader range of glycans [35–38]. Further categorization of VP8* binding specificities, and possible correlations with host-restriction and pathogenicity, are strongly needed. Consistent with a role in receptor binding and attachment to target cells, VP8* forms the two distal lobes of the rotavirus spike (Fig. 3Ai).

The remainder of the spike is formed by three copies of the VP5* proteolysis product (~ 60 kDa) and the extended N-terminal regions of VP8* [9,39]. The VP5* spike adopts a highly unusual conformation that has elements of both trimeric and dimeric symmetry (Fig. 3A). The trimeric, globular base of VP5* docks into a quasi-six-fold symmetric depression in the VP6 layer, such that each VP5* subunit approaches two VP6 trimers [9]. However, aside from general shape-complementarity, few interactions exist to stably anchor VP4 to the DLP. This observation is consistent with many aspects of VP4 biology, including a requirement for viral chaperone (NSP4)-mediated assembly and rapid release from the DLP upon VP7 dissociation [28,30,40].

Assembly of VP7 onto VP6 restricts dissociation of VP4 by constricting the diameter above the VP5* base (Fig. 3A) [9,26]. As the spike extends out of the virus particle, trimeric symmetry is broken. One of the VP5* subunits lies nearly flat to the particle surface (VP5*C), and the other two form dimeric interactions and project away from the virion [9,39]. The upright VP5* subunits bind the two VP8* lobes described above. The globular VP8* partner of VP5*C is presumed (but not definitively known) to dissociate from the virion after proteolysis, as it has not been observed in any rotavirus particle reconstruction. However, given the nature of the interactions known to exist between VP8* and VP5* at the tip of the spike (Fig. 3B), it is unlikely that a third VP8* could be present as its globular shape would sterically preclude VP5*C from adopting the conformation that is seen for it at the base of the spike.

Specific interactions with VP7 appear to be the key regulator of VP4 conformational changes during maturation and entry. Subtle asymmetry of the VP6 and VP7 layers suggests a mechanism driving VP5*C to adopt its unusual conformation at every identical position on the virion. A gap between two VP7 trimers enables specific interactions with the base of the VP5*C β-barrel domain (Fig. 3B) [9]. In contrast, the other VP7 trimers more closely surround the spike and are unavailable for interaction. Thus, the VP7 capsid, in part, dictates VP5* conformation after trypsin proteolysis. The function of VP4 maturation is thought to be analogous to that of enveloped virus fusion proteins, namely, to prime VP5* for refolding during membrane penetration [9,39,41]. During entry, VP7 uncoating releases constraints on the spike and permits VP5* to snap-back into a collapsed trimer structure [29,39]. This conformation of VP5* is considered a post-penetration state, analogous to the post-fusion state of enveloped virus fusion proteins [39,40].

Conclusions

High-resolution reconstruction of icosahedral viruses has become a rapid and reasonably facile approach to gain significant insights into virion functions, including assembly and entry [2,8,9]. The limitations of this technology, however, are illustrated in the paucity of information obtained for rotavirus VP3 and the pre-cleavage conformation of the VP4 spike. Cryo-EM tomography, which can image individual (non-averaged) virions [42], and novel methods of orienting particles [43], may be helpful in deriving additional data from “known” structures, and developing new hypotheses for mechanisms of virus biology.

Highlights.

  • Structures of rotavirus have greatly informed studies of replication and entry

  • The Reoviridae may have a conserved capsid-polymerase interaction mechanism

  • The rotavirus polymerase orientation suggests the location of the capping enzyme

  • The conformation of the rotavirus spike is regulated by the outer capsid protein

  • New methods are needed to study the rotavirus spike precursor and capping enzyme

Acknowledgments

The authors would like to thank Stephen Harrison and Ethan Settembre for helpful discussions and for sharing unpublished data. This work was supported by Public Health Service award Z01 AI000788 from the Intramural Research Program of the National Institutes of Allergy and Infectious Diseases, National Institutes of Health.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • **1.McClain B, Settembre E, Temple BR, Bellamy AR, Harrison SC. X-ray crystal structure of the rotavirus inner capsid particle at 3.8 A resolution. J Mol Biol. 2010;397:587–599. doi: 10.1016/j.jmb.2010.01.055. This study reported the atomic structure of the rotavirus double-layered particle, providing high resolution information on the organization of the VP2 and VP6 protein layers, the interface between the two, and the location of ligand channels for nucleotides and RNA. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Zhang X, Settembre E, Xu C, Dormitzer PR, Bellamy R, Harrison SC, Grigorieff N. Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. Proc Natl Acad Sci USA. 2008;105:1867–1872. doi: 10.1073/pnas.0711623105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Zientara S, Mertens PP, Stuart DI. The atomic structure of the bluetongue virus core. Nature. 1998;395:470–478. doi: 10.1038/26694. [DOI] [PubMed] [Google Scholar]
  • 4.Reinisch KM, Nibert ML, Harrison SC. Structure of the reovirus core at 3. 6 A resolution. Nature. 2000;404:960–967. doi: 10.1038/35010041. [DOI] [PubMed] [Google Scholar]
  • 5.Prasad BV, Rothnagel R, Zeng CQ, Jakana J, Lawton JA, Chiu W, Estes MK. Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus. Nature. 1996;382:471–473. doi: 10.1038/382471a0. [DOI] [PubMed] [Google Scholar]
  • 6.Mathieu M, Petitpas I, Navaza J, Lepault J, Kohli E, Pothier P, Prasad BV, Cohen J, Rey FA. Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion. EMBO J. 2001;20:1485–1497. doi: 10.1093/emboj/20.7.1485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Liemann S, Chandran K, Baker TS, Nibert ML, Harrison SC. Structure of the reovirus membrane-penetration protein, Mu1, in a complex with is protector protein, Sigma3. Cell. 2002;108:283–295. doi: 10.1016/s0092-8674(02)00612-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Zhang X, Jin L, Fang Q, Hui WH, Zhou ZH. 3. 3 A cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry. Cell. 2010;141:472–482. doi: 10.1016/j.cell.2010.03.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • **9.Settembre EC, Chen JZ, Dormitzer PR, Grigorieff N, Harrison SC. Atomic model of an infectious rotavirus particle. EMBO J. 2011;30:408–416. doi: 10.1038/emboj.2010.322. Using electron cryomicroscopy, the authors determined the structure of the entire rotavirus virion, and observed the unique, asymmetric conformation of the mature spike protein. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Aoki ST, Settembre EC, Trask SD, Greenberg HB, Harrison SC, Dormitzer PR. Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab. Science. 2009;324:1444–1447. doi: 10.1126/science.1170481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chen JZ, Settembre EC, Aoki ST, Zhang X, Bellamy AR, Dormitzer PR, Harrison SC, Grigorieff N. Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM. Proc Natl Acad Sci USA. 2009;106:10644–10648. doi: 10.1073/pnas.0904024106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lawton JA, Estes MK, Prasad BV. Three-dimensional visualization of mRNA release from actively transcribing rotavirus particles. Nat Struct Biol. 1997;4:118–121. doi: 10.1038/nsb0297-118. [DOI] [PubMed] [Google Scholar]
  • **13.Lu X, McDonald SM, Tortorici MA, Tao YJ, Vasquez-Del Carpio R, Nibert ML, Patton JT, Harrison SC. Mechanism for coordinated RNA packaging and genome replication by rotavirus polymerase VP1. Structure. 2008;16:1678–1688. doi: 10.1016/j.str.2008.09.006. Through X-ray crystallography, the authors determined the structure of the rotavirus RNA polymerase VP1 alone and in complex with RNA, allowing discovery of the mechanism by which the polymerase specifically recognizes viral templates. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Thouvenin E, Schoehn G, Rey F, Petitpas I, Mathieu M, Vaney MC, Cohen J, Kohli E, Pothier P, Hewat E. Antibody inhibition of the transcriptase activity of the rotavirus DLP: a structural view. J Mol Biol. 2001;307:161–172. doi: 10.1006/jmbi.2000.4479. [DOI] [PubMed] [Google Scholar]
  • 15.Zhang X, Walker SB, Chipman PR, Nibert ML, Baker TS. Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7. 6 A. Nat Struct Biol. 2003;10:1011–1018. doi: 10.1038/nsb1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • *16.Estrozi LF, Navaza J. Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way. J Struct Biol. 2010;172:253–260. doi: 10.1016/j.jsb.2010.06.023. Throught improvement of methodologies for processing single-particle 3D reconstruction data, the authors were able to define the position of the rotavirus polymerase VP1 within the virion core. [DOI] [PubMed] [Google Scholar]
  • **17.McDonald SM, Patton JT. Rotavirus VP2 core shell regions critical for viral polymerase activation. J Virol. 2011;85:3095–3105. doi: 10.1128/JVI.02360-10. Using a cell-free RNA synthesis system and purified recombinant rotavirus proteins, the authors were able to identify two functionally distinct regions of the VP2 capsid protein necessary for inducing viral RNA polymerase activity. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Zeng CQ, Estes MK, Charpilienne A, Cohen J. The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3. J Virol. 1998;72:201–208. doi: 10.1128/jvi.72.1.201-208.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.McDonald SM, Patton JT. Molecular characterization of a subgroup specificity associated with the rotavirus inner capsid protein VP2. J Virol. 2008;82:2752–2764. doi: 10.1128/JVI.02492-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chen D, Luongo CL, Nibert ML, Patton JT. Rotavirus open cores catalyze 5′-capping and methylation of exogenous RNA: evidence that VP3 is a methyltransferase. Virology. 1999;265:120–130. doi: 10.1006/viro.1999.0029. [DOI] [PubMed] [Google Scholar]
  • 21.Pizarro JL, Sandino AM, Pizarro JM, Fernandez J, Spencer E. Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3. J Gen Virol. 1991;72 (Pt 2):325–332. doi: 10.1099/0022-1317-72-2-325. [DOI] [PubMed] [Google Scholar]
  • 22.Sutton G, Grimes JM, Stuart DI, Roy P. Bluetongue virus VP4 is an RNA-capping assembly line. Nat Struct Mol Biol. 2007;14:449–451. doi: 10.1038/nsmb1225. [DOI] [PubMed] [Google Scholar]
  • 23.Ramadevi N, Burroughs NJ, Mertens PP, Jones IM, Roy P. Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus. Proc Natl Acad Sci USA. 1998;95:13537–13542. doi: 10.1073/pnas.95.23.13537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Martinez-Costas J, Sutton G, Ramadevi N, Roy P. Guanylyltransferase and RNA 5′-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus. J Mol Biol. 1998;280:859–866. doi: 10.1006/jmbi.1998.1926. [DOI] [PubMed] [Google Scholar]
  • 25.Lawton JA, Estes MK, Prasad BV. Identification and characterization of a transcription pause site in rotavirus. J Virol. 2001;75:1632–1642. doi: 10.1128/JVI.75.4.1632-1642.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Trask SD, Dormitzer PR. Assembly of highly infectious rotavirus particles recoated with recombinant outer capsid proteins. J Virol. 2006;80:11293–11304. doi: 10.1128/JVI.01346-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Poruchynsky MS, Atkinson PH. Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles. J Virol. 1991;65:4720–4727. doi: 10.1128/jvi.65.9.4720-4727.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Trask SD, Kim IS, Harrison SC, Dormitzer PR. A rotavirus spike protein conformational intermediate binds lipid bilayers. J Virol. 2010;84:1764–1770. doi: 10.1128/JVI.01682-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wolf M, Vo PT, Greenberg HB. Rhesus rotavirus entry into a polarized epithelium is endocytosis dependent and involves sequential VP4 conformational changes. J Virol. 2011;85:2492–2503. doi: 10.1128/JVI.02082-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Yoder JD, Trask SD, Vo TP, Binka M, Feng N, Harrison SC, Greenberg HB, Dormitzer PR. VP5* rearranges when rotavirus uncoats. J Virol. 2009;83:11372–11377. doi: 10.1128/JVI.01228-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Crawford SE, Mukherjee SK, Estes MK, Lawton JA, Shaw AL, Ramig RF, Prasad BV. Trypsin cleavage stabilizes the rotavirus VP4 spike. J Virol. 2001;75:6052–6061. doi: 10.1128/JVI.75.13.6052-6061.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Blanchard H, Yu X, Coulson BS, von Itzstein M. Insight into host cell carbohydrate-recognition by human and porcine rotavirus from crystal structures of the virion spike associated carbohydrate-binding domain (VP8*) J Mol Biol. 2007;367:1215–1226. doi: 10.1016/j.jmb.2007.01.028. [DOI] [PubMed] [Google Scholar]
  • 33.Dormitzer PR, Sun ZY, Wagner G, Harrison SC. The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site. EMBO J. 2002;21:885–897. doi: 10.1093/emboj/21.5.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Monnier N, Higo-Moriguchi K, Sun ZY, Prasad BV, Taniguchi K, Dormitzer PR. High-resolution molecular and antigen structure of the VP8* core of a sialic acid-independent human rotavirus strain. J Virol. 2006;80:1513–1523. doi: 10.1128/JVI.80.3.1513-1523.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Haselhorst T, Fleming FE, Dyason JC, Hartnell RD, Yu X, Holloway G, Santegoets K, Kiefel MJ, Blanchard H, Coulson BS, et al. Sialic acid dependence in rotavirus host cell invasion. Nat Chem Biol. 2009;5:91–93. doi: 10.1038/nchembio.134. [DOI] [PubMed] [Google Scholar]
  • 36.Huang P, Xia M, Tan M, Zhong W, Wei C, Wang L, Morrow A, Jiang X. Spike protein VP8* of human rotavirus recognizes HBGAs in a type-specific manner. J Virol. 2012 doi: 10.1128/JVI.05507-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Yu X, Coulson BS, Fleming FE, Dyason JC, von Itzstein M, Blanchard H. Novel structural insights into rotavirus recognition of ganglioside glycan receptors. J Mol Biol. 2011;413:929–939. doi: 10.1016/j.jmb.2011.09.005. [DOI] [PubMed] [Google Scholar]
  • **38.Hu L, Crawford SE, Czako R, Cortes-Penfield NW, Smith DF, Le Pendu J, Estes MK, Prasad BV. Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen. Nature. 2012 doi: 10.1038/nature10996. Using x-ray crystallography, the authors of this study determined the structure of a human rotavirus VP8* in complex with HBGA. These data provide a new model for the attachment of “sialic acid-independent” rotaviruses, which include the majority of those strains that infect and cause disease in humans. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Dormitzer PR, Nason EB, Prasad BV, Harrison SC. Structural rearrangements in the membrane penetration protein of a non-enveloped virus. Nature. 2004;430:1053–1058. doi: 10.1038/nature02836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • *40.Trask SD, McDonald SM, Patton JT. Structural insights into the coupling of virion assembly and rotavirus replication. Nat Rev Microbiol. 2012;10:165–177. doi: 10.1038/nrmicro2673. This comprehensive review focuses on structure-function analysis of several aspects of rotavirus replication, and expands upon some of the concepts presented in this article. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kim IS, Trask SD, Babyonyshev M, Dormitzer PR, Harrison SC. Effect of mutations in VP5 hydrophobic loops on rotavirus cell entry. J Virol. 2010;84:6200–6207. doi: 10.1128/JVI.02461-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Fontana J, Cardone G, Heymann JB, Winkler DC, Steven AC. Structural changes in influenza virus at low pH characterized by cryo-electron tomography. J Virol. 2012;86:2919–2929. doi: 10.1128/JVI.06698-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • *43.Wu W, Thomas JA, Cheng N, Black LW, Steven AC. Bubblegrams reveal the inner body of bacteriophage phiKZ. Science. 2012;335:182. doi: 10.1126/science.1214120. Using a novel controlled-damage technique during electron cryomicroscopy, the authors were able to determine the orientation of an asymmetric feature (the inner body) within an otherwise symmetric virion; this technique has clear implications for the capacity to determine the potentially asymmetric organization of the RNA and polymerase complexes within Reovirdae virions. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES