Controlling the surface charge of simple viruses

The vast majority of plant viruses are unenveloped, i.e., they lack a lipid bilayer that is characteristic of most animal viruses. The interactions between plant viruses, and between viruses and surfaces, properties that are essential for understanding their infectivity and to their use as bionanomaterials, are largely controlled by their surface charge, which depends on pH and ionic strength. They may also depend on the charge of their contents, i.e., of their genes or–in the instance of virus-like particles–encapsidated cargo such as nucleic acid molecules, nanoparticles or drugs. In the case of enveloped viruses, the surface charge of the capsid is equally important for controlling its interaction with the lipid bilayer that it acquires and loses upon leaving and entering host cells. We have previously investigated the charge on the unenveloped plant virus Cowpea Chlorotic Mottle Virus (CCMV) by measurements of its electrophoretic mobility. Here we examine the electrophoretic properties of a structurally and genetically closely related bromovirus, Brome Mosaic Virus (BMV), of its capsid protein, and of its empty viral shells, as functions of pH and ionic strength, and compare them with those of CCMV. From measurements of both solution and gel electrophoretic mobilities (EMs) we find that the isoelectric point (pI) of BMV (5.2) is significantly higher than that of CCMV (3.7), that virion EMs are essentially the same as those of the corresponding empty capsids, and that the same is true for the pIs of the virions and of their cleaved protein subunits. We discuss these results in terms of current theories of charged colloidal particles and relate them to biological processes and the role of surface charge in the design of new classes of drug and gene delivery systems.

Antiviral therapy in shrimp through plant virus VLP containing VP28 dsRNA against WSSV

The white spot syndrome virus (WSSV), currently affecting cultured shrimp, causes substantial economic losses to the worldwide shrimp industry. An antiviral therapy using double-stranded RNA interference (dsRNAi) by intramuscular injection (IM) has proven the most effective shrimp protection against WSSV. However, IM treatment is still not viable for shrimp farms. The challenge is to develop an efficient oral delivery system that manages to avoid the degradation of antiviral RNA molecules. The present work demonstrates that VLPs (virus-like particles) allow efficient delivery of dsRNAi as antiviral therapy in shrimp. In particular, VLPs derived from a virus that infects plants, such as cowpea chlorotic mottle virus (CCMV), in which the capsid protein (CP) encapsidates the dsRNA of 563 bp, are shown to silence the WSSV glycoprotein VP28 (dsRNAvp28). In experimental challenges in vivo, the VLPs- dsRNAvp28 protect shrimp against WSSV up to 40% by oral administration and 100% by IM. The novel research demonstrates that plant VLPs, which avoid zoonosis, can be applied to pathogen control in shrimp and also other organisms, widening the application window in nanomedicine.

Fluorescent Nanodiamonds Encapsulated by Cowpea Chlorotic Mottle Virus (CCMV) Proteins for Intracellular 3D-Trajectory Analysis

Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes within living cells. Fluorescent nanodiamond (ND) emitters provide cell compatibility and very high photostability. However, high stability, biocompatibility, and cellular uptake of these fluorescent NDs under physiological conditions are required for intracellular applications. Herein, highly stable NDs encapsulated with Cowpea chlorotic mottle virus capsid proteins (ND-CP) are prepared. A thin capsid protein layer is obtained around the NDs, which imparts reactive groups and high colloidal stability, while retaining the opto-magnetic properties of the coated NDs as well as the secondary structure of CPs adsorbed on the surface of NDs. In addition, the ND-CP shows excellent biocompatibility both in vitro and in vivo. Long-term 3D trajectories of the ND-CP with fine spatiotemporal resolutions are recorded; their intracellular motions are analyzed by different models, and the diffusion coefficient are calculated. The ND-CP with its brilliant optical properties and stability under physiological conditions provides us with a new tool to advance the understanding of cell biology, e.g., endocytosis, exocytosis, and active transport processes in living cells as well as intracellular dynamic parameters. <br>

Fluorescent Nanodiamonds Encapsulated by Cowpea Chlorotic Mottle Virus (CCMV) Proteins for Intracellular 3D-Trajectory Analysis

Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes within living cells. Fluorescent nanodiamond (ND) emitters provide cell compatibility and very high photostability. However, high stability, biocompatibility, and cellular uptake of these fluorescent NDs under physiological conditions are required for intracellular applications. Herein, highly stable NDs encapsulated with Cowpea chlorotic mottle virus capsid proteins (ND-CP) are prepared. A thin capsid protein layer is obtained around the NDs, which imparts reactive groups and high colloidal stability, while retaining the opto-magnetic properties of the coated NDs as well as the secondary structure of CPs adsorbed on the surface of NDs. In addition, the ND-CP shows excellent biocompatibility both in vitro and in vivo. Long-term 3D trajectories of the ND-CP with fine spatiotemporal resolutions are recorded; their intracellular motions are analyzed by different models, and the diffusion coefficient are calculated. The ND-CP with its brilliant optical properties and stability under physiological conditions provides us with a new tool to advance the understanding of cell biology, e.g., endocytosis, exocytosis, and active transport processes in living cells as well as intracellular dynamic parameters. <br>

Fluorescent Nanodiamonds Encapsulated by Cowpea Chlorotic Mottle Virus (CCMV) Proteins for Intracellular 3D-Trajectory Analysis

Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes within living cells. Fluorescent nanodiamond (ND) emitters provide cell...

Engineered protein cages for selective heparin encapsulation

A heparin-specific binding peptide was conjugated to a cowpea chlorotic mottle virus (CCMV) capsid protein, which was subsequently allowed to encapsulate heparin and form capsid-like protein cages. The encapsulation is...

Next generation sequencing as a tool in modern pest risk analysis: a case study of groundnuts (Arachis hypogaea) as a potential host of new viruses in western Kenya

Groundnut (Arachis hypogaea, L.) is grown in diverse environments throughout the semi-arid and sub-tropical regions of the world. Poor yields of 500-800kg/ha are attributed to poor agronomic practices, pests and diseases. The major disease reported in Kenya is Groundnut rosette disease (GRD). But recent observations in the field showed that the crop has varied and severe symptoms in addition to those caused by GRD. This required deeper analysis to establish the causal agents. Groundnut samples with virus-like symptoms were collected from western Kenya in 2016. Total RNA was extracted using All Prep RNA Mini Kit. Five mRNA libraries were prepared using the Illumina TrueSeq stranded mRNA library Prep Kit and pooled for multiplexed sequencing using an Illumina HiSeq 2500 to generate paired end reads (FastQ Sanger). The reads were analysed in the Galaxy project platform (customized). Quality reads were first mapped onto plant genome Refseq and unmapped reads isolated and mapped onto virus Refseq using Bowtie 2 (v2.2.3). Groundnut rosette virus satellite RNA, Groundnut rosette virus, Groundnut rosette assistor virus, Ethiopian tobacco bushy top virus, Cowpea polerovirus 2, Chickpea chlorotic stunt virus, Melon aphid-borne yellow virus, Phasey bean mild yellow virus, Beet mild yellowing virus, White clover mottle virus and Cotton leafroll dwarf virus were identified in four libraries. Other viruses (with less than 100 reads) including Bean common mosaic virus, Bean common mosaic necrosis virus, Cowpea chlorotic mottle virus RNA 3, Broad bean mottle virus RNA 3, Passion fruit woodiness virus among others were also mapped. Some of the viruses common in western Kenya were confirmed by PCR. The presence of at least three viruses in groundnuts in Western Kenya highlights the importance of starting a germplasm clean-up program of the plant material used as seed in this crop. Key words: Groundnuts, NGS, RefSeq, Viruses.