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Sorghum [Sorghum bicolor (L.) Moench.] is a versatile crop, grown in 30 countries and a food source for nearly 500 million people globally. Although the sorghum genome is sequenced, a limited understanding of gene function prevents the improvement of resistance against almost 150 species of viruses, bacteria, fungus, and parasitic plants to improve productivity. Here, we present a Brome mosaic virus (BMV)-based virus-induced gene silencing (VIGS) to silence target genes for functional study in sorghum. This protocol achieves 100% sorghum infection with BMV by growing the plants at 18 °C instead of 22 °C. Using this method, one can achieve gene silencing in sorghum up to 100% of the inoculated plants.

Protein-based nanoreactors are generated by encapsulating an enzyme inside the capsid of the cowpea chlorotic mottle virus (CCMV). Here, three different noncovalent methods are described to efficiently incorporate enzymes inside the capsid of these viral protein cages. The methods are based on pH, leucine zippers, and electrostatic interactions respectively, as a driving force for encapsulation. The methods are exclusively described for the enzymes horseradish peroxidase, glucose oxidase, and Pseudozyma antarctica lipase B, but they are also applicable for other enzymes.

Viral protein cage-based nanoreactors can be generated by encapsulation of catalytic metal nanoparticles within the capsid structure. In this method, coat proteins of the cowpea chlorotic mottle virus (CCMV) are used to sequester gold nanoparticles (Au NPs) in buffered solutions at neutral pH to form CCMV-Au hybrid nanoparticles. This chapter describes detailed methods for the encapsulation of Au NPs into CCMV protein cages. Protocols for the reduction of nitroarenes by using CCMV-Au NPs as catalyst are described as an example for the catalytic activity of Au NPs in the protein cages.

The controlled self-assembly of protein cages is vital for the use of these nanocompartments in biomedical and nanotechnological applications. Recently, we showed that by combining different structural peptide elements, it is possible to assemble viral capsid proteins in distinct well-defined morphologies. In this chapter, a triblock copolypeptide is discussed, consisting of a metal ion-coordinating hexahistidine tag, a stimulus-responsive elastin-like polypeptide and a pH-responsive self-assembling viral capsid protein. This protein is able to form two different types of capsids, depending on the assembly pathway that is followed. Here, we focus on the metal ion-induced assembly process and describe the relevant experimental procedures to induce and utilize this assembly behavior.

We report a protocol for efficient cell-free synthesis of cowpea chlorotic mottle virus (CCMV)-like particles containing a broad range of lengths and sequences of RNA. Our protocol starts with a purified stock of wild-type CCMV (protocols for harvesting and purifying the virus are detailed elsewhere) and features three basic steps: disassembly of the CCMV and purification of the capsid protein (CP) from the viral RNA; coassembly of the purified CP and an RNA of choice; and characterization of the assembly products. We highlight several key factors that increase the yield of the assembly reaction: the CP should be uncleaved and sufficiently free of viral RNA; the length of the RNA should be between about 100 and 4000 nucleotides; and the stoichiometry of CP and RNA should be 6-1 by mass. Additionally, we point out that separating the assembly reaction into multiple steps-by successively lowering the ionic strength and then the pH of the assembly buffers-results in the highest yields of well-formed, nuclease-resistant, CCMV-like particles. Finally, we describe methods for characterizing the assembly products using native agarose gel electrophoresis and negative-stain transmission electron microscopy.

The DNA origami technique is a widely used method to create customized, complex, spatially well-defined two-dimensional (2D) and three-dimensional (3D) DNA nanostructures. These structures have huge potential to serve as smart drug-delivery vehicles and molecular devices in various nanomedical and biotechnological applications. However, so far only little is known about the behavior of these novel structures in living organisms or in cell culture/tissue models. Moreover, enhancing pharmacokinetic bioavailability and transfection properties of such structures still remains a challenge. One intriguing approach to overcome these issues is to coat DNA origami nanostructures with proteins or lipid membranes. Here, we show how cowpea chlorotic mottle virus (CCMV) capsid proteins (CPs) can be used for coating DNA origami nanostructures. We present a method for disassembling native CCMV particles and isolating the pure CP dimers, which can further bind and encapsulate a rectangular DNA origami shape. Owing to the highly programmable nature of DNA origami, packaging of DNA nanostructures into viral protein cages could find imminent uses in enhanced targeting and cellular delivery of various active nano-objects, such as enzymes and drug molecules.

In cellular systems, compartmentalization plays an important role in the protection and regulation of enzymes. Controlled encapsulation of enzymes in nanocompartments is crucial in understanding biocatalytic processes in the cellular environment. We have recently described an enzymatic method to covalently attach enzymes, equipped with a small recognition peptide, to the interior of viral capsids. Viral capsids are especially interesting in this respect, as they form very well-defined nanoparticles with a uniform size and shape. Here, we describe the relevant experimental procedures to encapsulate a model enzyme into the interior of a viral capsid, purify the resulting viral capsids, and measure the catalytic activity of the encapsulated enzymes.

The self-assembly of virus-like particles may lead to materials which combine the unique characteristics of viruses, such as precise size control and responsivity to environmental cues, with the properties of abiotic cargo. For a few different viruses, shell proteins are amenable to the in vitro encapsulation of non-genomic cargo in a regular protein cage. In this chapter we describe protocols of high-efficiency in vitro self-assembly around functionalized gold nanoparticles for three examples of icosahedral and non-icosahedral viral protein cages derived from a plant virus, an animal virus, and a human retrovirus. These protocols can be readily adapted with small modifications to work for a broad variety of inorganic and organic nanoparticles.

Northern blotting is a valuable method for detection and quantification of RNA in the field of virology. Although many methods including a various versions of polymerase chain reaction have been developed over the years, Northern blotting has been still considered as a useful and effective method for the analysis of progeny RNA accumulation for viral and subviral pathogens, such as satellite RNAs, in plant hosts. Here, we describe a detailed Northern blot protocol for efficient detection and quantification of viral and satellite RNAs from plant hosts.

Analysis of viral RNA encapsidation assay provides a rapid means of assaying which of the progeny RNA are competent for packaging into stable mature virions. Generally, a parallel analysis of total RNA and RNA obtained from purified virions is advisable for accurate interpretation of the results. In this, we describe a series of in vivo assays in which viral RNA encapsidation can be verified. These include whole plants inoculated either mechanically or by Agroinfiltration and protoplasts. The encapsidation assay described here is for an extensively studied plant RNA virus, brome mosaic virus, and can be reliably applied to other viral systems as well as with appropriate buffers. In principle, the encapsidation assay requires purification of virions from either symptomatic leaves or transfected plant protoplasts followed by RNA isolation. The procedure involves grinding the infected tissue in an appropriate buffer followed by a low speed centrifugation step to remove the cell debris. The supernatant is then emulsified with an organic solvent such as chloroform to remove chlorophyll and cellular material. After a low seed centrifugation, the supernatant is subjected to high speed centrifugation to concentrate the virus as a pellet. Depending on the purity required, the partially purified virus preparation is further subjected to sucrose density gradient centrifugation. Following purification of virions, encapsidated RNA is isolated using standard phenol-chloroform extraction procedure. An important step in the encapsidation assay is the comparative analysis of total and virion RNA preparations by Northern hybridization. This would allow the investigator to compare the number of progeny RNA components synthesized during replication vs. encapsidation. Northern blots are normally hybridized with radioactively labeled RNA probes (riboprobes) for specific and sensitive detection of desired RNA species.

The interaction between viral polymerases and their cognate RNAs is vital to regulate the timing and abundance of viral replication products. Despite this, only minimal detailed information is available for the interaction between viral polymerases and cognate RNAs. We study the biochemical interactions using two viral polymerases that could serve as models for other plus-strand RNA viruses: the replicase from the tripartite brome mosaic virus (BMV), and the recombinant RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV). Replicase binding sites in the BMV RNAs were mapped using a template competition assay. The minimal length of RNA required for RNA binding by the HCV RdRp was determined using fluorescence spectroscopy. Lastly, regions of the HCV RdRp that contact the RNA were determined by a method coupling reversible protein-RNA crosslinking, affinity purification, and mass spectrometry. These analyses of RdRp-RNA interaction will be presented as three topics in this chapter.

Virus-induced gene silencing (VIGS) is a powerful RNA-silencing based technology adapted for the study of host-gene function. VIGS functions through the expression of a host gene from a virus vector. Both the virus-encoded host sequence and the homologous host target messenger RNA are destroyed or made inactive through a host surveillance system. Here, we describe procedures for the use of a new virus vector for VIGS in monocotyledonous hosts and, in particular, in rice (Oryza sativa), a species for which no VIGS vector was previously available.