Atomic Force Microscopy of Viruses.

Atomic force microscopy employs a nanometric tip located at the end of a micro-cantilever to probe surface-mounted samples at nanometer resolution. Because the technique can also work in a liquid environment it offers unique possibilities to study individual viruses under conditions that mimic their natural milieu. Here, we review how AFM imaging can be used to study the surface structure of viruses including that of viruses lacking a well-defined symmetry. Beyond imaging, AFM enables the manipulation of single viruses by force spectroscopy experiments. Pulling experiments can provide information about the early events of virus-host interaction between the viral fibers and the cell membrane receptors. Pushing experiments measure the mechanical response of the viral capsid and its contents and can be used to show how virus maturation and exposure to different pH values change the mechanical response of the viruses and the interaction between the capsid and genome. Finally, we discuss how studying capsid rupture and self-healing events offers insight in virus uncoating pathways.

Built-in mechanical stress in viral shells.

Mechanical properties of biological molecular aggregates are essential to their function. A remarkable example are double-stranded DNA viruses such as the φ29 bacteriophage, that not only has to withstand pressures of tens of atmospheres exerted by the confined DNA, but also uses this stored elastic energy during DNA translocation into the host. Here we show that empty prolated φ29 bacteriophage proheads exhibit an intriguing anisotropic stiffness which behaves counterintuitively different from standard continuum elasticity predictions. By using atomic force microscopy, we find that the φ29 shells are approximately two-times stiffer along the short than along the long axis. This result can be attributed to the existence of a residual stress, a hypothesis that we confirm by coarse-grained simulations. This built-in stress of the virus prohead could be a strategy to provide extra mechanical strength to withstand the DNA compaction during and after packing and a variety of extracellular conditions, such as osmotic shocks or dehydration.

Durable protein lattices of clathrin that can be functionalized with nanoparticles and active biomolecules.

Biological molecules that self-assemble and interact with other molecules are attractive building blocks for engineering biological devices. DNA has been widely used for the creation of nanomaterials, but the use of proteins remains largely unexplored. Here, we show that clathrin can form homogeneous and extended two-dimensional lattices on a variety of substrates, including glass, metal, carbon and plastic. Clathrin is a three-legged protein complex with unique self-assembling properties and is relevant in the formation of membrane transport vesicles in eukaryotic cells. We used a fragment of the adaptor protein epsin to immobilize clathrin lattices on the substrates. The lattices span multiple square millimetres with a regular periodicity of 30 nm and can be functionalized via modified subunits of clathrin with either inorganic nanoparticles or active enzymes. The lattices can be stored for months after crosslinking and stabilization with uranyl acetate. They could be dehydrated and rehydrated without loss of function, offering potential applications in sensing and as biosynthetic reactors.

Structural and mechanical study of a self-assembling protein nanotube.

We report a structural characterization of self-assembling nanostructures. Using atomic force microscopy (AFM), we discovered that partially hydrolyzed alpha-lactalbumin organizes in a 10-start helix forming tubes with diameters of only 21 nm. We probed the mechanical strength of these nanotubes by locally indenting them with an AFM tip. To extract the material properties of the nanotubes, we modeled the experiment using finite element methods. Our study shows that artificial helical protein self-assembly can yield very stable, strong structures that can function either as a model system for artificial self-assembly or as a nanostructure with potential for practical applications.

DNA-mediated anisotropic mechanical reinforcement of a virus.

In this work, we provide evidence of a mechanism to reinforce the strength of an icosahedral virus by using its genomic DNA as a structural element. The mechanical properties of individual empty capsids and DNA-containing virions of the minute virus of mice are investigated by using atomic force microscopy. The stiffness of the empty capsid is found to be isotropic. Remarkably, the presence of the DNA inside the virion leads to an anisotropic reinforcement of the virus stiffness by approximately 3%, 40%, and 140% along the fivefold, threefold, and twofold symmetry axes, respectively. A finite element model of the virus indicates that this anisotropic mechanical reinforcement is due to DNA stretches bound to 60 concavities of the capsid. These results, together with evidence of biologically relevant conformational rearrangements of the capsid around pores located at the fivefold symmetry axes, suggest that the bound DNA may reinforce the overall stiffness of the viral particle without canceling the conformational changes needed for its infectivity.

Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication.

Practical components for three-dimensional molecular nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nanometers on a side, that can self-assemble in seconds with near-quantitative yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an atomic force microscope, we have measured the axial compressibility of DNA and observed the buckling of the double helix under high loads.

Persistence and transmission of natural type I feline coronavirus infection.

To examine the mode of natural transmission and persistence of feline coronavirus (FCoV), FCoV strains shed by domestic cats were investigated over periods of up to 7 years. An RT-PCR that amplified part of the 3' end of the viral spike (S) gene was devised to distinguish FCoV types I and II. All but 1 of 28 strains of FCoV from 43 cats were type I. Nucleotide identities of the amplified 320 bp product from 49 type I FCoVs ranged from 79 to 100 %. The consensus partial S sequence of isolates recovered from persistently infected cats at time intervals spanning years was generally conserved. While most cats were infected with a single strain, a few may have been infected by more than one strain. Cats that were transiently infected and ceased shedding could be re-infected with either the same, or a different, strain. In most cases, whether a cat became persistently or transiently infected was independent of the virus strain. However, one strain was unusual in that it infected the majority of cats in the household simultaneously and was still being shed 18 months later. Factors that influence whether FCoV establishes lifelong infection in some cats and not others are determined mainly by the host response to infection.

Deformation and collapse of microtubules on the nanometer scale.

We probe the local mechanical properties of microtubules at the nanometer scale by radial indentation with a scanning force microscope tip. We find a linear elastic regime that can be described by both thin-shell theory and finite element methods, in which microtubules are modeled as hollow tubes. We also find a nonlinear regime and catastrophic collapse of the microtubules under large loads. The main physics of protein shells at the nanometer scale shows simultaneously aspects of continuum elasticity in their linear response, as well as molecular graininess in their nonlinear behavior.