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Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

A high-resolution structure of the bacteriophage MS2 sheds light on the structure of the genome and how the genome is delivered into a bacterium. Atomic structure of an ssRNA genome Hong Zhou and colleagues provide the first description of genome–capsid interactions in a spherical single-stranded RNA (ssRNA) virus, using the bacteriophage MS2 as a model. Unlike double-stranded DNA viruses that pump their genome into a preformed capsid, ssRNA viruses co-assemble their capsid with their genome. Here the authors determine the MS2 structure at 3.6 Å resolution and are able to trace around 80% of the backbone of the viral genome, identifying regions that react with the maturation protein and providing insights into the ssRNA capsid co-assembly process. Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid 1 , 2 , 3 , single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome 4 , 5 , 6 , 7 ; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’ (F-pilus) 8 ; it was the first fully sequenced organism 9 and is a model system for studies of translational gene regulation 10 , 11 , RNA–protein interactions 12 , 13 , 14 , and RNA virus assembly 15 , 16 , 17 . Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T  = 3 icosahedral lattice 18 , 19 . The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection 8 , but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′ end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly. MS2 is a single-stranded RNA bacteriophage that infects its host via adsorption to bacterial pili. Here the authors visualize the MS2 virion with asymmetric cryo-EM reconstruction, revealing that the genome of MS2 adopts a specific structure of asymmetrically distributed stem-loops connected to the capsid.

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.

Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20–30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA–protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein–protein interactions, consistent with the RNA–protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.

Background The RNA bacteriophage MS2 is an RNA phage that infects the bacterium E. coli and is one of the most studied and prototypical model phages in molecular biology and microbiology. Previous research revealed complex translational control and fine-tuning for MS2 replication. However, the dynamics of its transcriptional activity and replication during the life cycles within the bacteria remain elusive. Methods Here, we employed Nanopore Direct RNA sequencing (DRS) to investigate the transcriptome and epitranscriptome landscape of the MS2 in infected E. coli throughout multiple life cycles. Results We discovered that MS2 phages sustain a high level of transcriptional activity required for replication. We found large amounts of subgenomic small transcripts from RNA degradation, Nanopore DRS bias, and transcripts containing the coat -encoding region, required for virion assembly. We found the error-prone activity of the MS2 replicase produced hybrid reads from the template-switching mechanism. We finally evidenced that RNA modification is conserved throughout the entire life cycle in full-length transcripts without the acquisition of new modifications, whereas small transcripts did acquire newly modified sites. The conserved sequence and secondary structure (U-rich hairpin) of Ψ installation sites were the most amenable to RNA modification, from potentially the host RluA-mediated installation. Conclusions Overall, our investigation revealed a more complex transcriptional dynamics of MS2 phages than anticipated within E. coli to maintain its growth and replication under host pressure.

Abstract Cheater viruses cannot replicate on their own yet replicate faster than the wild type (WT) when the 2 viruses coinfect the same cell. Cheaters must possess dual genetic features: a defect, which leads to their inability to infect cells on their own, and a selective advantage over WT during coinfection. Previously, we have discovered 2 point-mutant cheaters of the MS2 bacteriophage. Here, we set out to discover the possible repertoire of cheater MS2 viruses by performing experimental evolution at a very high multiplicity of infection. Our results revealed a third point-mutant cheater that arose in 8 biological replicas. Each of the 3 primary cheaters disrupts the fine balance necessary for phage replication, in different ways that create a defect + advantage. We found that over time, the point-mutant cheaters accumulate additional secondary mutations, which alter other stages of the viral replication cycle, complementing the disruptions created by the original cheater. Intriguingly, cheater and secondary mutations almost always reside in very close proximity on the genome. This region encodes for multiple functions: overlapping reading frames as well as overlapping RNA structures critical for transitioning from one stage to another in the viral replication cycle. This region of overlap explains the dual functions of cheaters, as one mutation can have pleiotropic effects. Overall, these findings underscore how viruses, whose dense genomes often have overlapping functions, can easily evolve point-mutant cheaters, and how cheaters can evolve to alter the intricate balance of the viral replication cycle.

Single-stranded RNA bacteriophages (ssRNA phages) infect Gram-negative bacteria via a single maturation protein (Mat), which attaches to a retractile pilus of the host. Here we present structures of the ssRNA phage MS2 in complex with the Escherichia coli F-pilus, showing a network of hydrophobic and electrostatic interactions at the Mat-pilus interface. Moreover, binding of the pilus induces slight orientational variations of the Mat relative to the rest of the phage capsid, priming the Mat-connected genomic RNA (gRNA) for its release from the virions. The exposed tip of the attached Mat points opposite to the direction of the pilus retraction, which may facilitate the translocation of the gRNA from the capsid into the host cytosol. In addition, our structures determine the orientation of the assembled F-pilin subunits relative to the cell envelope, providing insights into the F-like type IV secretion systems. Single-stranded RNA bacteriophages use a single maturation protein (Mat) to attach to a retractile pilus of the bacterial host. Here, the authors report the structures of the MS2 phage bound to the host receptor F-pili and define the orientations of Mat relative to the cell and emanating F-pili, providing new insights into the F-like type IV secretion systems.

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then theMat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.

We report a theoretical investigation of the electrohydrodynamic properties of spherical soft particles composed of permeable concentric layers that differ in thickness, soft material density, chemical composition, and flow penetration degree. Starting from a recent numerical scheme developed for the computation of the direct-current electrophoretic mobility (μ) of diffuse soft bioparticles, the dependence of μ on the electrolyte concentration and solution pH is evaluated taking the known three-layered structure of bacteriophage MS2 as a supporting model system (bulk RNA, RNA-protein bound layer, and coat protein). The electrokinetic results are discussed for various layer thicknesses, hydrodynamic flow penetration degrees, and chemical compositions, and are discussed on the basis of the equilibrium electrostatic potential and hydrodynamic flow field profiles that develop within and around the structured particle. This study allows for identifying the cases where the electrophoretic mobility is a function of the inner structural and chemical specificity of the particle and not only of its outer surface properties. Along these lines, we demonstrate the general inapplicability of the notions of zeta potential (ζ) and surface charge for quantitatively interpreting electrokinetic data collected for such systems. We further shed some light on the physical meaning of the isoelectric point. In particular, numerical and analytical simulations performed on structured soft layers in indifferent electrolytic solution demonstrate that the isoelectric point is a complex ionic strength-dependent signature of the flow permeation properties and of the chemical and structural details of the particle. Finally, the electrophoretic mobilities of the MS2 virus measured at various ionic strength levels and pH values are interpreted on the basis of the theoretical formalism aforementioned. It is shown that the electrokinetic features of MS2 are to a large extent determined not only by the external proteic capsid but also by the chemical composition and hydrodynamic flow permeation of/within the inner RNA-protein bound layer and bulk RNA part of the bacteriophage. The impact of virus aggregation, as revealed by decreasing diffusion coefficients for decreasing pH values, is also discussed.