Increased Clinical Signs and Mortality in IFNAR(-/-) Mice Immunised with the Bluetongue Virus Outer-Capsid Proteins VP2 or VP5, after Challenge with an Attenuated Heterologous Serotype.

Bluetongue is an economically important disease of domesticated and wild ruminants caused by bluetongue virus (BTV). There are at least 36 different serotypes of BTV (the identity of which is determined by its outer-capsid protein VP2), most of which are transmitted by Culicoides biting midges. IFNAR(-/-) mice immunised with plant-expressed outer-capsid protein VP2 (rVP2) of BTV serotypes -1, -4 or -8, or the smaller outer-capsid protein rVP5 of BTV-10, or mock-immunised with PBS, were subsequently challenged with virulent strains of BTV-4 or BTV-8, or with an attenuated clone of BTV-1 (BTV-1RGC7). The mice that had received rVP2 generated a protective immune response against the homologous BTV serotype, reducing viraemia (as detected by qRT-PCR), the severity of clinical signs and mortality levels. No cross-serotype protection was observed after challenge with the heterologous BTV serotypes. However, the severity of clinical signs, viraemia and fatality levels after challenge with the attenuated strain of BTV-1 were all increased in mice immunised with rVP2 of BTV-4 and BTV-8, or with rVP5 of BTV10. The possibility is discussed that non-neutralising antibodies, reflecting serological relationships between the outer-capsid proteins of these different BTV serotypes, could lead to 'antibody-dependent enhancement of infection' (ADE). Such interactions could affect the epidemiology and emergence of different BTV strains in the field and would therefore be relevant to the design and implementation of vaccination campaigns.

The Tobacco Mosaic Virus Origin of Assembly Sequence is Dispensable for Specific Viral RNA Encapsidation but Necessary for Initiating Assembly at a Single Site.

We have investigated whether the presence of the origin of assembly sequence (OAS) of tobacco mosaic virus (TMV) is necessary for the specific encapsidation of replicating viral RNA. To this end TMV coat protein was expressed from replicating RNA constructs with or without the OAS in planta. In both cases the replicating RNA was specifically encapsidated to give nucleoprotein nanorods, though the yield in the absence of the OAS was reduced to about 60% of that in its presence. Moreover, the nanorods generated in the absence of the OAS were more heterogeneous in length and contained frequent structural discontinuities. These results strongly suggest that the function of the OAS is to provide a unique site for the initiation of viral assembly, leading to a one-start helix, rather than the selection of virus RNA for packaging.

Designer-length palladium nanowires can be templated by the central channel of tobacco mosaic virus nanorods.

We have developed methods for the templated synthesis of palladium nanowires (Pd NWs) within the central channel of tobacco mosaic virus (TMV) nanorods of various lengths. We show that uniform 4 nm diameter Pd NWs can be produced by selective growth within these channels by including the capping reagent, poly(vinyl-pyrrolidone) (PVP30K) and reducing the metal precursor to metallic palladium with ascorbic acid. The length of the Pd NWs can be controlled either by varying the length of the nanorod templates and/or through alterations to the reaction conditions. We have also demonstrated bimetallic gold (Au)-palladium (Pd) in-situ metallization of TMV nanorods resulting in the production of Pd NWs 6 nm gold nanoparticles attached to their ends. The materials produced have many potential applications in the construction of nanoscale devices.

The Use of a Replicating Virus Vector For in Planta Generation of Tobacco Mosaic Virus Nanorods Suitable For Metallization.

The production of designer-length tobacco mosaic virus (TMV) nanorods in plants has been problematic in terms of yields, particularly when modified coat protein subunits are incorporated. To address this, we have investigated the use of a replicating potato virus X-based vector (pEff) to express defined length nanorods containing either wild-type or modified versions of the TMV coat protein. This system has previously been shown to be an efficient method for producing virus-like particles of filamentous plant viruses. The length of the resulting TMV nanorods can be controlled by varying the length of the encapsidated RNA. Nanorod lengths were analyzed with a custom-written Python computer script coupled with the Nanorod UI user interface script, thereby generating histograms of particle length. In addition, nanorod variants were produced by incorporating coat protein subunits presenting metal-binding peptides at their C-termini. We demonstrate the utility of this approach by generating nanorods that bind colloidal gold nanoparticles.

Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants.

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence.

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the Begomovirus genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.

Investigating the Biological Relevance of In Vitro-Identified Putative Packaging Signals at the 5' Terminus of Satellite Tobacco Necrosis Virus 1 Genomic RNA.

Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that >90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only <1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA.IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite tobacco necrosis virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules.

The 3.3 Å structure of a plant geminivirus using cryo-EM.

Geminiviruses are major plant pathogens that threaten food security globally. They have a unique architecture built from two incomplete icosahedral particles, fused to form a geminate capsid. However, despite their importance to agricultural economies and fundamental biological interest, the details of how this is realized in 3D remain unknown. Here we report the structure of Ageratum yellow vein virus at 3.3 Å resolution, using single-particle cryo-electron microscopy, together with an atomic model that shows that the N-terminus of the single capsid protein (CP) adopts three different conformations essential for building the interface between geminate halves. Our map also contains density for ~7 bases of single-stranded DNA bound to each CP, and we show that the interactions between the genome and CPs are different at the interface than in the rest of the capsid. With additional mutagenesis data, this suggests a central role for DNA binding-induced conformational change in directing the assembly of geminate capsids.

Production of Mosaic Turnip Crinkle Virus-Like Particles Derived by Coinfiltration of Wild-Type and Modified Forms of Virus Coat Protein in Plants.

When the coat protein reading frame of turnip crinkle virus (TCV) is transiently expressed in leaves, virus-like particles (VLPs) are readily formed. However, after introducing genetic modifications to the full-length coat protein sequence, such as the introduction of an epitope-specific sequence within the coat protein sequence or the in-frame carboxyl terminal fusion of GFP, the formation of such modified VLPs is poor. However, by coexpression of one of these modified forms with wild-type TCV coat protein by the coinfiltration of appropriate Agrobacterium suspensions, VLP generation is enhanced through the formation of "mosaics," that is, individual VLPs consisting of both modified and wild-type subunits (also known as phenotypically mixed VLPs). Here we describe methods for the introduction of genetic modifications into the TCV coat protein sequence, the production of mosaic TCV VLPs and their characterization.

In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires.

We have utilized plant-based transient expression to produce tobacco mosaic virus (TMV)-based nano-rods of predetermined lengths. This is achieved by expressing RNAs containing the TMV origin of assembly sequence (OAS) and the sequence of the TMV coat protein either on the same RNA molecule or on two separate constructs. We show that the length of the resulting nano-rods is dependent upon the length of the RNA that possesses the OAS element. By expressing a version of the TMV coat protein that incorporates a metal-binding peptide at its C-terminus in the presence of RNA containing the OAS we have been able to produce nano-rods of predetermined length that are coated with cobalt-platinum. These nano-rods have the properties of defined-length nano-wires that make them ideal for many developing bionanotechnological processes.

Synthetic plant virology for nanobiotechnology and nanomedicine.

Nanotechnology is a rapidly expanding field seeking to utilize nano-scale structures for a wide range of applications. Biologically derived nanostructures, such as viruses and virus-like particles (VLPs), provide excellent platforms for functionalization due to their physical and chemical properties. Plant viruses, and VLPs derived from them, have been used extensively in biotechnology. They have been characterized in detail over several decades and have desirable properties including high yields, robustness, and ease of purification. Through modifications to viral surfaces, either interior or exterior, plant-virus-derived nanoparticles have been shown to support a range of functions of potential interest to medicine and nano-technology. In this review we highlight recent and influential achievements in the use of plant virus particles as vehicles for diverse functions: from delivery of anticancer compounds, to targeted bioimaging, vaccine production to nanowire formation. WIREs Nanomed Nanobiotechnol 2017, 9:e1447. doi: 10.1002/wnan.1447 For further resources related to this article, please visit the WIREs website.

The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein.

Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation.

Genetic engineering and characterization of Cowpea mosaic virus empty virus-like particles.

The development of methods for the production of empty Cowpea mosaic virus (CPMV) virus-like particles (VLPs) that are devoid of RNA, eVLPs, has renewed promise in CPMV capsid technologies. The recombinant nature of CPMV eVLP production means that the extent and variety of genetic modifications that may be incorporated into the particles is theoretically much greater than those that can be made to infectious CPMV virions due to restrictions on viral propagation of the latter. Free of the infectious agent, the genomic RNA, these particles are now finding potential uses in vaccine development, in vivo imaging, drug delivery, and other nanotechnology applications that make use of internal loading of the empty particles. Here we describe methods for the genetic modification and production of CPMV eVLPs and describe techniques useful for their characterization.

Exploiting plant virus-derived components to achieve in planta expression and for templates for synthetic biology applications.

This review discusses the varying roles that have been played by many plant-viral regulatory sequences and proteins in the creation of plant-based expression systems and virus particles for use in nanotechnology. Essentially, there are two ways of expressing an exogenous protein: the creation of transgenic plants possessing a stably integrated gene construction, or the transient expression of the desired gene following the infiltration of the gene construct. Both depend on disarmed strains of Agrobacterium tumefaciens to deliver the created gene construction into cell nuclei, usually through the deployment of virus-derived components. The importance of efficient mRNA translation in the latter process is highlighted. Plant viruses replicate to sustain an infection to promote their survival. The major product of this, the virus particle, is finding increasing roles in the emerging field of bionanotechnology. One of the major products of plant-viral expression is the virus-like particle (VLP). These are increasingly playing a role in vaccine development. Similarly, many VLPs are suitable for the investigation of the many facets of the emerging field of synthetic biology, which encompasses the design and construction of new biological functions and systems not found in nature. Genetic and chemical modifications to plant-generated VLPs serve as ideal starter templates for many downstream synthetic biology applications.

Isolation of an asymmetric RNA uncoating intermediate for a single-stranded RNA plant virus.

We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.

Top 10 plant viruses in molecular plant pathology.

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

Peptide-controlled access to the interior surface of empty virus nanoparticles.

The structure of Cowpea mosaic virus (CPMV) is known to high resolution, thereby enabling the rational use of the particles in diverse applications, from vaccine design to nanotechnology. A recently devised method for the production of empty virus-like particles (eVLPs) has opened up new possibilities for CPMV capsid-based technologies, such as internal mineralisation of the particle. We have investigated the role of the carboxyl (C) terminus of the small coat (S) protein in controlling access to the interior of CPMV eVLPs by determining the efficiency of internal mineralisation. The presence of the C-terminal 24-amino acid peptide of the S protein was found to inhibit internal mineralisation, an effect that could be eliminated by enzymatic removal of this region. We have also demonstrated the amenability of the C terminus to genetic modification. Substitution with six histidine residues generated stable particles and facilitated external mineralisation by cobalt. These findings demonstrate consistent internal and external mineralisation of CPMV, and will aid the further exploration and development of the use of eVLPs for bionanotechnological and medical applications.

Recent advances of cowpea mosaic virus-based particle technology.

Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as a means of presenting peptides for vaccine purposes. However, the existing technology has limitations in regard to the size and nature of the peptides which can be presented and has problems regarding bio-containment. Recent developments suggest ways by which these problems can be overcome, increasing the range of potential applications of CPMV-based particle technology.

Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless.

Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N. benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.