An official website of the United States government
Abstract Satellite tobacco mosaic virus (STMV) is a model system for studying viral assembly and stability due to its architecture: a single-stranded RNA genome enclosed in an icosahedral capsid. Coupling a polarizable force-field to enhanced sampling, we explored at high-resolution the long-timescale structural dynamics of a complete ∼1M-atom STMV. RNA-free capsids exhibit remarkable stability at physiological salt concentrations, suggesting an evolutionary adaptation for capsid reuse during the viral life cycle. This observation challenges the notion that empty capsids are exclusively products of abortive assembly, positioning them instead as functional intermediates in viral reproduction. Additionally, RNA encapsidation creates electrostatic dependencies that magnesium ions mitigate, stabilizing both RNA and capsid through long-residence-time interactions with phosphate groups. Chloride ions further influence capsid permeability by modulating salt-bridge disruptions and interprotomeric interactions, with these effects being pH-dependent: enhanced at pH < 7, preserving nucleocapsid integrity, or weakened at pH = 7, facilitating disassembly and RNA release.
more »
« less
RNA-induced Allosteric Coupling Drives Viral Capsid Assembly
Understanding the mechanisms by which single-stranded RNA viruses regulate capsid assembly around their RNA genomes has become increasingly important for the development of both antiviral treatments and drug delivery systems. In this study, we investigate the effects of RNA-induced allostery in a single-stranded RNA virus—Levivirus bacteriophage MS2 assembly—using the computational methods of the Dynamic Flexibility Index and the Dynamic Coupling Index. We demonstrate that not only does asymmetric binding of RNA to a symmetric MS2 coat protein dimer increase the flexibility of the distant FG-loop, inducing a conformational change to an asymmetric dimer, but also RNA binding reorganizes long-distance communications, making all the other positions extremely sensitive to the fluctuation of the ordered FG-loop. Additionally, we find that a point mutation in the FG-loop, W82R, leads to the loss of this asymmetry in communications, likely being a leading cause for assembly-deficient dimers. Lastly, this dominant communication that enhances its dynamic coupling with all the distal positions is not only a property of the dimer but is also exhibited by all the observed capsid intermediates. This strong dynamic coupling allows for unidirectional signal transduction that drives the formation of the experimentally observed capsid intermediates and fully assembled capsid.
more »
« less
- Award ID(s):
- 2239518
- PAR ID:
- 10565957
- Publisher / Repository:
- APS
- Date Published:
- Journal Name:
- PRX Life
- Volume:
- 2
- Issue:
- 1
- ISSN:
- 2835-8279
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content—specifically, RNAs 3 and 4—assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific “packaging signals” throughout the viral RNA to package their monopartite genomes.more » « less
-
The coat proteins (CPs) of single-stranded RNA bacteriophages (ssRNA phages) directly assemble around the genomic RNA (gRNA) to form a near-icosahedral capsid with a single maturation protein (Mat) that binds the gRNA and interacts with the retractile pilus during infection of the host. Understanding the assembly of ssRNA phages is essential for their use in biotechnology, such as RNA protection and delivery. Here, we present the complete gRNA model of the ssRNA phage Qβ, revealing that the 3′ untranslated region binds to the Mat and the 4127 nucleotides fold domain-by-domain, and is connected through long-range RNA–RNA interactions, such as kissing loops. Thirty-three operator-like RNA stem-loops are located and primarily interact with the asymmetric A/B CP-dimers, suggesting a pathway for the assembly of the virions. Additionally, we have discovered various forms of the virus-like particles (VLPs), including the canonical T = 3 icosahedral, larger T = 4 icosahedral, prolate, oblate forms, and a small prolate form elongated along the 3-fold axis. These particles are all produced during a normal infection, as well as when overexpressing the CPs. When overexpressing the shorter RNA fragments encoding only the CPs, we observed an increased percentage of the smaller VLPs, which may be sufficient to encapsidate a shorter RNA.more » « less
-
Abstract We investigated the relationship between mutations and dynamics inEscherichia colidihydrofolate reductase (DHFR) using computational methods. Our study focused on the M20 and FG loops, which are known to be functionally important and affected by mutations distal to the loops. We used molecular dynamics simulations and developed position‐specific metrics, including the dynamic flexibility index (DFI) and dynamic coupling index (DCI), to analyze the dynamics of wild‐type DHFR and compared our results with existing deep mutational scanning data. Our analysis showed a statistically significant association between DFI and mutational tolerance of the DHFR positions, indicating that DFI can predict functionally beneficial or detrimental substitutions. We also applied an asymmetric version of our DCI metric (DCIasym) to DHFR and found that certain distal residues control the dynamics of the M20 and FG loops, whereas others are controlled by them. Residues that are suggested to control the M20 and FG loops by our DCIasymmetric are evolutionarily nonconserved; mutations at these sites can enhance enzyme activity. On the other hand, residues controlled by the loops are mostly deleterious to function when mutated and are also evolutionary conserved. Our results suggest that dynamics‐based metrics can identify residues that explain the relationship between mutation and protein function or can be targeted to rationally engineer enzymes with enhanced activity.more » « less
-
Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA–protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)—for which RNA–protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA–protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA–protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA–protein interactions, while the growth process is driven less by RNA–protein interactions and more by protein–protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1103/PRXLife.2.013012
