Location of the dsRNA-Dependent Polymerase, VP1, in Rotavirus Particles (original) (raw)
Related papers
Journal of …, 2008
Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T ؍ 13 to T ؍ 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.
Journal of …, 2008
Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T ؍ 13 to T ؍ 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.
Biological research, 1994
The structural relationship between VP6 (inner capsid polypeptide) and the viral core was studied using chemical cross-linking with dithiobis(succinimidyl propionate). Crosslinked single shelled and reconstituted rotavirus particles, suggest the existence of a complex organization of VP6 molecules in the inner capsid and a direct interaction with the core polypeptide VP3. The inhibition of the recovery of RNA polymerase activity associated with the reconstitution of the single shelled particle in the presence of antiVP6 monoclonal antibodies indicates that a VP6 domain between amino acids 56 and 58 seems to be important in viral transcription. A VP6 gene temperature-sensitive mutant (ts G) carrying a mutation affecting assembly of single shelled particles was used in reconstitution experiments. The mutant was able to recover RNA polymerase activity at restrictive temperature. Wild type cores or VP6 were able to reconstitute the particle with both the mutant cores and VP6. These resu...
Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1
Structure, 2008
Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3 0 end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 Å resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus l3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3 0 sequence. Well-defined interactions with these bases position the RNA so that its 3 0 end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3 0 end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.
Journal of Virology, 2011
To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (؉RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3 end of rotavirus ؉RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral ؉RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with ؉RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of ؉RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with ؉RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates.
Structure, 2003
The capsid shells of these viruses, however, exhibit striking architectural differences. Except for the single-Baylor College of Medicine Houston, Texas 77030 shelled cypoviruses such as the cytoplasmic polyhedrosis virus (CPV), all other viruses in the Reoviridae have 3 State Key Lab for Biocontrol Institute of Entomology additional protein shells, such as the double-shelled rice dwarf virus (RDV) (Lu et al., 1998), and triple-shelled Zhongshan University Guangzhou 510275 rotavirus (Shaw et al., 1993) and bluetongue virus (BTV) (Grimes et al., 1998). In addition to conferring host speci-China ficity and mediating cell entry, these additional layers are believed to play important structural roles in maintaining the stability of the thin inner shell and sequestering the Summary dsRNA genome (Lawton et al., 2000). The inner shells of the Reoviridae are more homogenous and can be The single-shelled cytoplasmic polyhedrosis virus divided into two major groups. Those in the first group (CPV) is a unique member of the Reoviridae. Despite have a smooth inner shell made up of 120 CSP molecules lacking protective outer shells, it exhibits striking capenclosed by one or two outer T ϭ 13 layers, as exemplisid stability and is capable of endogenous RNA tranfied by BTV, RDV, and rotavirus. Those in the second scription and processing. The 8 Å three-dimensional group also have an inner shell consisting of 120 CSP structure of CPV by electron cryomicroscopy reveals molecules, but this shell is decorated by turrets (the secondary structure elements present in the capsid mRNA capping complexes) on the icosahedral vertices proteins CSP, LPP, and TP, which have ␣ϩ folds. The and by molecular clamps (large protrusions) joining extensive nonequivalent interactions between CSP neighboring CSP molecules. In addition, these viruses and LPP, the unique CSP protrusion domain, and the either have incomplete outer T ϭ 13 layers (e.g., orthoperfect inter-CSP surface complementarities may acreovirus [Dryden et al., 1993; Reinisch et al., 2000] and count for the enhanced capsid stability. The slanted aquareovirus [Shaw et al., 1996]) or completely lack any disposition of TP functional domains and the stacking outer protein layer (e.g., CPV [Hill et al., 1999; Xia et of channel constrictions suggest an iris diaphragmal., 2003; Zhang et al., 1999]). In these viruses, mRNA like mechanism for opening/closing capsid pores and transcription and posttranscriptional processing take turret channels in regulating the highly coordinated place in a series of well-coordinated steps, beginning steps of mRNA transcription, processing, and release. with mRNA transcription at the transcriptional enzyme complexes underneath the vertices of the inner shell, Introduction followed by 5Ј end mRNA capping and subsequent release through the multifunctional turret (Bartlett et al., RNA transcription is a fundamental process involving a 1974; Bellamy and Harvey, 1976; Furuichi, 1974; Furuichi series of well-coordinated processes catalyzed by multiet al., 1976; Reinisch et al., 2000; White and Zweerink, functional enzymes, often embedded in multicompo-1976; Xia et al., 2003; Yazaki and Miura, 1980; Zhang et nent macromolecular complexes. Double-stranded (ds) al., 1999). RNA viruses in the family Reoviridae are extreme exam-Having only a single shell, CPV is structurally the simples of such multifunctional RNA transcriptional maplest member of the Reoviridae. Despite lacking the chines. Their hosts include plants, insects, mammals, outer protective layers existing in other dsRNA viruses, and humans, and their structural proteins have little to CPV virions are resistant to chemical treatments, includno recognizable sequence homologies (reviewed by ing cations, high pH, trypsin, chymotrypsin, ribo-Mertens et al., 2000). Still, viruses in the nine genera of nuclease A, deoxyribonuclease, phospholipase, and this family all contain a characteristic segmented dsRNA SDS, and retain infectivity for weeks at Ϫ15ЊC to 25ЊC genome and a highly conserved dsRNA-dependent sin-(Mertens et al., 2000; Zhang et al., 2002). The relative gle-stranded RNA polymerase enclosed in a capsid shell simplicity and unusual stability of CPV make it an attracmade up of 120 molecules of the inner capsid shell tive system for studying the structural basis of RNA protein (CSP) (reviewed by Lawton et al., 2000; Nibert transcription and posttranscriptional processing. While and Schiff, 2001; Patton and Spencer, 2000). The Reovirits infection of silkworms can have a negative economic idae are all capable of endogenous mRNA transcription impact in Asia, CPV is also recognized as an emerging within an intact virus particle, using viral-encoded enbiocontrol agent, serving as an environmentally friendly zymes for transcription initiation, elongation, 5Ј capping, pesticide for fruit and vegetable farming (Mertens et al., 2000). Previous low resolution electron cryomicroscopy (cryoEM) structures showed that CPV shares similar *Correspondence: z.h.zhou@uth.tmc.edu
Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus
Nature
Characterising the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. In dsDNA and dsRNA viruses, the packaged double-stranded nucleic acid, constrained by the rigidity of the doublehelix, adopts a liquid crystalline arrangement 1-5. Owing to the different cellular fates of DNA and RNA, the life cycles of these viruses are strikingly dissimilar. Current models suggest that dsDNA viruses predominantly display single-spooled conformations due to the lack of genome segmentation and the absence of intracapsid transcriptional machinery 6-8. As dsRNA triggers host defence mechanisms if released into the cytoplasm 9 , dsRNA viruses retain their genomes within a core particle containing the enzymes required for RNA replication and transcription 10,11,12. Their genomes vary greatly in the degree of segmentation. In reoviruses (Reoviridae, 10-12 segments) the genome organizes in nonspooled fashion and is tightly associated with the viral RNA-dependent RNA polymerases (RdRPs) 11-14. However, whether this organization is generally applicable in dsRNA viruses remains unknown. Here, we use cryogenic electron microscopy (cryo-EM) to show that dsRNA viruses
Subunit Folds and Maturation Pathway of a dsRNA Virus Capsid
Structure, 2013
The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. f6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an a-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.