The effect of genome length on ejection forces in bacteriophage lambda

A variety of viruses tightly pack their genetic material into protein capsids that are barely large enough to enclose the genome. In particular, in bacteriophages, forces as high as 60 pN are encountered during packaging and ejection, produced by DNA bending elasticity and self-interactions. The high forces are believed to be important for the ejection process, though the extent of their involvement is not yet clear. As a result, there is a need for quantitative models and experiments that reveal the nature of the forces relevant to DNA ejection. Here we report measurements of the ejection forces for two different mutants of bacteriophage lambda, lambda b221cI26 and lambda cI60, which differ in genome length by ~30%. As expected for a force-driven ejection mechanism, the osmotic pressure at which DNA release is completely inhibited varies with the genome length: we find inhibition pressures of 15 atm and 25 atm, respectively, values that are in agreement with our theoretical calculations

Viral RNAs are unusually compact.

A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly

Genome organization and interaction with capsid protein in a multipartite RNA virus.

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

Reconstituted plant viral capsids can release genes to mammalian cells

AbstractThe nucleocapsids of many plant viruses are significantly more robust and protective of their RNA contents than those of enveloped animal viruses. In particular, the capsid protein (CP) of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is of special interest because it has been shown to spontaneously package, with high efficiency, a large range of lengths and sequences of single-stranded RNA molecules. In this work we demonstrate that hybrid virus-like particles, assembled in vitro from CCMV CP and a heterologous RNA derived from a mammalian virus (Sindbis), are capable of releasing their RNA in the cytoplasm of mammalian cells. This result establishes the first step in the use of plant viral capsids as vectors for gene delivery and expression in mammalian cells. Furthermore, the CCMV capsid protects the packaged RNA against nuclease degradation and serves as a robust external scaffold with many possibilities for further functionalization and cell targeting

Statistics of Shear-Induced Rearrangements in a Two-Dimensional Model Foam

Under steady shear, a foam relaxes stress through intermittent rearrangements of bubbles accompanied by sudden drops in the stored elastic energy. We use a simple model of foam that incorporates both elasticity and dissipation to study the statistics of bubble rearrangements in terms of energy drops, the number of nearest neighbor changes, and the rate of neighbor-switching (T1) events. We do this for a two-dimensional system as a function of system size, shear rate, dissipation mechanism, and gas area fraction. We find that for dry foams, there is a well-defined quasistatic limit at low shear rates where localized rearrangements occur at a constant rate per unit strain, independent of both system size and dissipation mechanism. These results are in good qualitative agreement with experiments on two-dimensional and three-dimensional foams. In contrast, we find for progessively wetter foams that the event size distribution broadens into a power law that is cut off only by system size. This is consistent with criticality at the melting transition

Boojums and the Shapes of Domains in Monolayer Films

Domains in Langmuir monolayers support a texture that is the two-dimensional version of the feature known as a boojum. Such a texture has a quantifiable effect on the shape of the domain with which it is associated. The most noticeable consequence is a cusp-like feature on the domain boundary. We report the results of an experimental and theoretical investigation of the shape of a domain in a Langmuir monolayer. A further aspect of the investigation is the study of the shape of a ``bubble'' of gas-like phase in such a monolayer. This structure supports a texture having the form of an inverse boojum. The distortion of a bubble resulting from this texture is also studied. The correspondence between theory and experiment, while not perfect, indicates that a qualitative understanding of the relationship between textures and domain shapes has been achieved.Comment: replaced with published version, 10 pages, 13 figures include

Folding Langmuir Monolayers

The maximum pressure a two-dimensional surfactant monolayer is able to withstand is limited by the collapse instability towards formation of three-dimensional material. We propose a new description for reversible collapse based on a mathematical analogy between the formation of folds in surfactant monolayers and the formation of Griffith Cracks in solid plates under stress. The description, which is tested in a combined microscopy and rheology study of the collapse of a single-phase Langmuir monolayer of 2-hydroxy-tetracosanoic acid (2-OH TCA), provides a connection between the in-plane rheology of LM's and reversible folding

Chiral symmetry breaking in Langmuir monolayers and smectic films

Langmuir monolayers and freely suspended smectic films can exhibit a spontaneous breaking of chiral symmetry. The order parameter that characterizes this symmetry breaking is coupled to variations in the direction of molecular tilt. As a result, chiral symmetry breaking leads to the spontaneous formation of complex equilibrium patterns with either 1D or 2D modifications in the direction of molecular tilt. A Landau theory for this pattern formation gives a general phase diagram, which includes a uniform nonchiral phase, a striped pattern, a square lattice, and a uniform chiral phase

A Simple RNA-DNA Scaffold Templates the Assembly of Monofunctional Virus-Like Particles

Using the components of a particularly well-studied plant virus, cowpea chlorotic mottle virus (CCMV), we demonstrate the synthesis of virus-like particles (VLPs) with one end of the packaged RNA extending out of the capsid and into the surrounding solution. This construct breaks the otherwise perfect symmetry of the capsid and provides a straightforward route for monofunctionalizing VLPs using the principles of DNA nanotechnology. It also allows physical manipulation of the packaged RNA, a previously inaccessible part of the viral architecture. Our synthesis does not involve covalent chemistry of any kind; rather, we trigger capsid assembly on a scaffold of viral RNA that is hybridized at one end to a complementary DNA strand. Interaction of CCMV capsid protein with this RNA-DNA template leads to selective packaging of the RNA portion into a well-formed capsid but leaves the hybridized portion poking out of the capsid through a small hole. We show that the nucleic acid protruding from the capsid is capable of binding free DNA strands and DNA-functionalized colloidal particles. Separately, we show that the RNA-DNA scaffold can be used to nucleate virus formation on a DNA-functionalized surface. We believe this self-assembly strategy can be adapted to viruses other than CCMV.Engineering and Applied SciencesPhysic

Two-Stage Dynamics of In Vivo Bacteriophage Genome Ejection

Biopolymer translocation is a key step in viral infection processes. The transfer of information-encoding genomes allows viruses to reprogram the cell fate of their hosts. Constituting 96% of all known bacterial viruses [A. Fokine and M. G. Rossmann, Molecular architecture of tailed double-stranded DNA phages, Bacteriophage 4, e28281 (2014)], the tailed bacteriophages deliver their DNA into host cells via an “ejection” process, leaving their protein shells outside of the bacteria; a similar scenario occurs for mammalian viruses like herpes, where the DNA genome is ejected into the nucleus of host cells, while the viral capsid remains bound outside to a nuclear-pore complex. In light of previous experimental measurements of in vivo bacteriophage λ ejection, we analyze here the physical processes that give rise to the observed dynamics. We propose that, after an initial phase driven by self-repulsion of DNA in the capsid, the ejection is driven by anomalous diffusion of phage DNA in the crowded bacterial cytoplasm. We expect that this two-step mechanism is general for phages that operate by pressure-driven ejection, and we discuss predictions of our theory to be tested in future experiments