Keith Saunders

Mutational analysis of complementary-sense genes of African cassava mosaic virus DNA A

We have investigated the ability of African cassava mosaic virus DNA A mutants, containing disrupted complementary-sense genes, to infect Nicotiana benthamiana and to replicate in Nicotiana tabacum protoplasts. Three overlapping open reading frames (ORFs) with the capacity to encode proteins with an Mr greater than 10K (AC1, AC2 and AC3) are highly conserved between geminiviruses that infect dicotyledonous plants and one (AC4) is less well conserved. Of these, only AC1 is a prerequisite for DNA replication; disruption of this ORF rendered the DNA noninfectious in plants and prevented DNA replication in protoplasts. Disruption of ORF AC2 prevented plant infection but mutants were capable of autonomous replication and replicated DNA B in trans in protoplasts to produce DNA forms that comigrated with wild-type virus DNAs. The AC2 mutant phenotype suggests that the product of this ORF is involved in virus spread within the plant. Mutants in which ORF AC3 had been disrupted retained the ability to replicate and to infect plants systemically although symptom development was delayed and attenuated, and mutant DNA accumulated to much lower levels (10 to 20%) in comparison with wild-type infection. Typical geminate virus particles were observed in extracts of plants infected with ORF AC3 mutants indicating that this gene is not essential for coat protein synthesis or virus assembly but possibly acts by modulating virus levels in infected tissues. Disruption of ORF AC4 had no effect on infectivity or symptom development suggesting that this ORF is maintained only because it overlaps the highly conserved ORF AC1.

Genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed.

A full-length copy of a single genomic component of the whitefly-transmitted geminivirus ageratum yellow vein virus (AYVV) has been cloned from an extract of infected Ageratum conyzoides originating from Singapore. Sequence analysis shows that the genomic component encodes two virion-sense (V1 and V2) and four complementary-sense open reading frames (C1-C4), typical of DNA A of whitefly-transmitted geminiviruses from the Eastern hemisphere. A genomic component equivalent to DNA B was not detected in extracts of infected A. conyzoides. The cloned genomic component produced a systemic infection in Nicotiana benthamiana, Phaseolus vulgaris and Lycopersicon esculentum when introduced into plants by agroinoculation, and symptoms were identical to those produced by wild-type virus introduced into these hosts using viruliferous whiteflies. However, attempts to re-establish a systemic infection in A. conyzoides either by agroinoculation or by whitefly transmission of the cloned progeny were unsuccessful, suggesting that additional factors are required for infection of the natural host. The significance of A. conyzoides as a reservoir host for the economically important geminivirus diseases is discussed.

Efficient generation of cowpea mosaic virus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants.

To elucidate the mechanism of formation of cowpea mosaic virus (CPMV) particles, RNA-2-encoded precursor proteins were expressed in Spodoptera frugiperda cells. Processing of the 105K and 95K polyproteins in trans to give the mature Large (L) and Small (S) coat proteins required both the 32K proteinase cofactor and the 24K proteinase itself, while processing of VP60, consisting of the fused L-S protein, required only the 24K proteinase. Release of the L and S proteins resulted in the formation of virus-like particles (VLPs), showing that VP60 can act as a precursor of virus capsids. Processing of VP60 expressed in plants also led to efficient production of VLPs. Analysis of the VLPs produced by the action of the 24K proteinase on precursors showed that they were empty (RNA-free). This has important implications for the use of CPMV VLPs in biotechnology and nanotechnology as it will permit the use of noninfectious particles.

Replication promiscuity of DNA-β satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-β satellite localizes sequences involved in replication

Pseudorecombination studies in Nicotiana benthamiana demonstrate that Ageratum yellow vein virus (AYVV) and Eupatorium yellow vein virus (EpYVV) can functionally interact with DNA-beta satellites associated with AYVV, EpYVV, cotton leaf curl Multan virus (CLCuMV) and honeysuckle yellow vein virus (HYVV). In contrast, CLCuMV shows some specificity in its ability to interact with distinct satellites and HYVV is able to interact only with its own satellite. Using an N. benthamiana leaf disk assay, we have demonstrated that HYVV is unable to trans-replicate other satellites. To investigate the basis of trans-replication compatibility, deletion mutagenesis of AYVV DNA-beta has been used to localize the origin of replication to approximately 360 nt, encompassing the ubiquitous nonanucleotide/stem-loop structure, satellite conserved region (SCR) and part of the intergenic region immediately upstream of the SCR. Additional deletions within this intergenic region have identified a region that is essential for replication. The capacity for DNA-beta satellites to functionally interact with distinct geminivirus species and its implications for disease diversification are discussed.

Adaptation from whitefly to leafhopper transmission of an autonomously replicating nanovirus-like DNA component associated with ageratum yellow vein disease

Ageratum yellow vein disease is caused by the whitefly-transmitted monopartite begomovirus Ageratum yellow vein virus and a DNA beta satellite component. Naturally occurring symptomatic plants also contain an autonomously replicating nanovirus-like DNA 1 component that relies on the begomovirus and DNA beta for systemic spread and whitefly transmission but is not required for maintenance of the disease. Here, we show that systemic movement of DNA 1 occurs in Nicotiana benthamiana when co-inoculated with the bipartite begomovirus Tomato golden mosaic virus and the curtovirus Beet curly top virus (BCTV), but not with the mastrevirus Bean yellow dwarf virus. BCTV also mediates the systemic movement of DNA 1 in sugar beet, and the nanovirus-like component is transmitted between plants by the BCTV leafhopper vector Circulifer tenellus. We also describe a second nanovirus-like component, referred to as DNA 2, that has only 47% nucleotide sequence identity with DNA 1. The diversity and adaptation of nanovirus components are discussed.

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.

COMPLEMENTATION OF AFRICAN CASSAVA MOSAIC VIRUS AC2 GENE FUNCTION IN A MIXED BIPARTITE GEMINIVIRUS INFECTION

We have previously demonstrated that African cassava mosaic virus (ACMV) DNAs A and B efficiently complement the systemic spread of tomato golden mosaic virus (TGMV) DNA A when co-agroinoculated onto Nicotiana benthamiana. Here, we show that a mixture of an ACMV DNA A AC2 mutant and DNA B that is normally unable to systemically infect N. benthamiana can do so at low frequency when co-agroinoculated with TGMV DNA A. Analysis of viral DNA showed that the AC2 mutation was retained during infection. The mixture of genomic components was sap transmissible, indicating that systemic infectivity is not specifically attributable to the use of agroinoculation. In the presence of TGMV DNA A, ACMV coat protein as well as the DNA B gene products BV1 and BC1 were detected in systemically infected tissues. The results demonstrate that dysfunctional AC2 can be complemented in planta by its TGMV homologue AL2.

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.

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

Summary 16 I. I. 16 II. II. 18 III. III. 20 IV. IV. 23 Acknowledgements 24 References 24 Summary 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.

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.