Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

Trans-Activator Binding Site Context in RCNMV Modulates Subgenomic mRNA Transcription.

Many positive-sense RNA viruses transcribe subgenomic (sg) mRNAs during infections that template the translation of a subset of viral proteins. Red clover necrotic mosaic virus (RCNMV) expresses its capsid protein through the transcription of a sg mRNA from RNA1 genome segment. This transcription event is activated by an RNA structure formed by base pairing between a trans-activator (TA) in RNA2 and a trans-activator binding site (TABS) in RNA1. In this study, the impact of the structural context of the TABS in RNA1 on the TA-TABS interaction and sg mRNA transcription was investigated using in vitro and in vivo approaches. The results (i) generated RNA secondary structure models for the TA and TABS, (ii) revealed that the TABS is partially base paired with proximal upstream sequences, which limits TA access, (iii) demonstrated that the aforementioned intra-RNA1 base pairing involving the TABS modulates the TA-TABS interaction in vitro and sg mRNA levels during infections, and (iv) revealed that the TABS in RNA1 can be modified to mediate sg mRNA transcription in a TA-independent manner. These findings advance our understanding of transcriptional regulation in RCNMV and provide novel insights into the origin of the TA-TABS interaction.

Plant Virus-Based Nanoparticles for the Delivery of Agronomic Compounds as a Suspension Concentrate.

Nanoparticle formulations of agrichemicals may enhance their performance while simultaneously mitigating any adverse environmental effects. Red clover necrotic mosaic virus (RCNMV) is a soil-transmitted plant virus with many inherent attributes that allow it to function as a plant virus-based nanoparticle (PVN) when loaded with biologically active ingredients. Here we describe how to formulate a PVN loaded with the nematicide abamectin (Abm) beginning with the propagation of the virus through the formulation, deactivation, and characterization of the finished product.

In Vitro-Reassembled Plant Virus-Like Particles of Hibiscus Chlorotic Ringspot Virus (HCRSV) as Nano-Protein Cages for Drugs.

Spherical shaped plant viruses require a precise quantity, size, and shape of their coat protein subunits to assemble into virions of identical dimensions. The capsid of spherical plant virus particles typically consists of a precisely shaped protein cage, which in many cases is assembled from identical coat protein subunits. In addition to packaging the viral genome, such protein cages may have the capacity to load foreign compounds, either large molecules (e.g., polymers) or small molecules (e.g., anticancer chemotherapy drugs). Therefore, reassembled protein cages of suitable viruses can serve as carriers for cargo loading, which is what makes them an attractive platform for drug delivery. Here we describe methods to reassemble plant virus-like particles of hibiscus chlorotic ringspot virus (HCRSV) as nano-protein cages including the techniques to purify coat protein, prepare virus-like particles, and load them with foreign compounds.

Generation of recombinant protein shells of Johnson grass chlorotic stripe mosaic virus in tobacco plants and their use as drug carrier.

The development and use of virus-like particles (VLPs) is a growing field with a powerful potential in generation of nanoparticles. In the present study we have attempted to generate and use empty shells of Johnson grass chlorotic stripe mosaic virus (JgCSMV, a member of the genus Aureusvirus, family Tombusviridae) as VLP nanoparticles for drug loading. In order to successfully produce recombinant JgCSMV-derived VLPs, we followed an approach based on cloning of the JgCSMV CP gene into pBI121 vector and introduction of the latter into Agrobacterium rhizogenes and transformation of tobacco cells for coat protein expression. Expression in tobacco tissue was demonstrated in transformed hairy roots as a model system. Recombinant VLPs were purified, analyzed by immune assay and visulalized by electron microscopy. Next, we explored the possibility of using JgCSMV-derived VLPs as a nanocontainer for loading the anticancer drug doxorubicin (DOX), taking advantage of the reversible swelling of VLPs in vitro. The results showed that transformed hairy roots produced high levels of the recombinant protein that readily assembled to form empty shells with overall structure similar to native virus particles. In addition, we demonstrated that JgCSMV-VLPs could function as vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first research addressing the question of how this icosahedral virus (JgCSMV) can be used for the production of nanocontainers for biomedical applications.

Protein expression strategies in Tobacco necrosis virus-D.

Tobacco necrosis virus (TNV-D) has a plus-strand RNA genome that is neither 5' capped nor 3' poly-adenylated. Instead, it utilizes a 3' cap-independent translational enhancer (3'CITE) located in its 3' untranslated region (UTR) for translation of its proteins. We have examined the protein expression strategies used by TNV-D and our results indicate that: (i) a base pairing interaction between conserved ACCA and UGGU motifs in the genomic 5'UTR and 3'CITE, respectively, is not required for efficient plant cell infection, (ii) similar potential 5'UTR-3'CITE interactions in the two viral subgenomic mRNAs are not needed for efficient translation of viral proteins in vitro, (iii) a small amount of capsid protein is translated from the viral genome by a largely 3'CITE-independent mechanism, (iv) the larger of two possible forms of capsid protein is efficiently translated, and (v) p7b is translated from subgenomic mRNA1 by a leaky scanning mechanism.

Insight into the three-dimensional structure of maize chlorotic mottle virus revealed by Cryo-EM single particle analysis.

Maize chlorotic mottle virus (MCMV) is the only member of the Machlomovirus genus in the family Tombusviridae. Here, we obtained the Cryo-EM structure of MCMV by single particle analysis with most local resolution at approximately 4 Å. The Cα backbone was built based on residues with bulky side chains. The resolved C-terminus of the capsid protein subunit and obvious openings at the 2-fold axis demonstrated the compactness of the asymmetric unit, which indicates an important role in the stability of MCMV. The Asp116 residue from each subunit around the 5-fold and 3-fold axes contributed to the negative charges in the centers of the pentamers and hexamers, which might serve as a solid barrier against the leakage of genomic RNA. Finally, the loops most exposed on the surface were analyzed and are proposed to be potential functional sites related to MCMV transmission.

Loading and release mechanism of red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin.

Loading and release mechanisms of Red clover necrotic mosaicvirus (RCNMV) derived plant viral nanoparticle (PVN) are shown for controlled delivery of the anticancer drug, doxorubicin (Dox). Previous studies demonstrate that RCNMV's structure and unique response to divalent cation depletion and re-addition enables Dox infusion to the viral capsid through a pore formation mechanism. However, by controlling the net charge of RCNMV outer surface and accessibility of RCNMV interior cavity, tunable release of PVN is possible via manipulation of the Dox loading capacity and binding locations (external surface-binding or internal capsid-encapsulation) with the RCNMV capsid. Bimodal release kinetics is achieved via a rapid release of surface-Dox followed by a slow release of encapsulated Dox. Moreover, the rate of Dox release and the amount of released Dox increases with an increase in environmental pH or a decrease in concentration of divalent cations. This pH-responsive Dox release from PVN is controlled by Fickian diffusion kinetics where the release rate is dependent on the location of the bound or loaded active molecule. In summary, controllable release of Dox-loaded PVNs is imparted by 1) formulation conditions and 2) driven by the capsid's pH- and ion- responsive functions in a given environment.

Construction and characterization of an aureusvirus defective RNA.

Defective RNAs (D RNAs) are small RNA replicons derived from viral RNA genomes. No D RNAs have been found associated with members of the plus-strand RNA virus genus Aureusvirus (family Tombusviridae). Accordingly, we sought to construct a D RNA for the aureusvirus Cucumber leaf spot virus (CLSV) using the known structure of tombusvirus defective interfering RNAs as a guide. An efficiently accumulating CLSV D RNA was generated that contained four non-contiguous regions of the viral genome and this replicon was used as a tool to studying viral cis-acting RNA elements. The results of structural and functional analyses indicated that CLSV contains counterparts for several of the major RNA elements found in tombusviruses. However, although similar, the CLSV D RNA and its components are distinct and provide insights into RNA-based specificity and mechanisms of function.

Translational readthrough in Tobacco necrosis virus-D.

The plus-strand RNA genome of Tobacco necrosis virus-D (TNV-D) expresses its polymerase via translational readthrough. The RNA signals involved in this readthrough process were characterized in vitro using a wheat germ extract translation system and in vivo via protoplast infections. The results indicate that (i) TNV-D requires a long-range RNA-RNA interaction between an extended stem-loop (SL) structure proximal to the readthrough site and a sequence in the 3'-untranslated region of its genome; (ii) stability of the extended SL structure is important for its function; (iii) TNV-D readthrough elements are compatible with UAG and UGA, but not UAA; (iv) a readthrough defect can be rescued by a heterologous readthrough element in vitro, but not in vivo; and (v) readthrough elements can also mediate translational frameshifting. These results provide new information on determinants of readthrough in TNV-D and further support the concept of a common general mechanism for readthrough in Tombusviridae.

Predicting the loading of virus-like particles with fluorescent proteins.

The virus-like particle (VLP) of the Cowpea Chlorotic Mottle Virus (CCMV) has often been used to encapsulate foreign cargo. Here we show two different rational design approaches, covalent and noncovalent, for loading teal fluorescent proteins (TFP) into the VLP. The covalent loading approach allows us to gain control over capsid loading on a molecular level. The achieved loading control is used to accurately predict the loading of cargo into CCMV VLP. The effects of molecular confinement were compared for the differently loaded VLPs created with the covalent method. We see that the loading of more than 10 fluorescent proteins in the 18 nm internal cavity of the CCMV capsid gives rise to a maximum efficiency of homo-FRET between the loaded proteins, as measured by fluorescence anisotropy. This shows that already at low levels of VLP loading molecular crowding starts to play a role.

New insight into the structure of RNA in red clover necrotic mosaic virus and the role of divalent cations revealed by small-angle neutron scattering.

Red clover necrotic mosaic virus (RCNMV) is a 36-nm-diameter, T = 3 icosahedral plant virus with a genome that is split between two single-stranded RNA molecules of approximately 3.9 kb and 1.5 kb, as well as a 400-nucleotide degradation product. The structure of the virus capsid and its response to removing Ca(2+) and Mg(2+) was previously studied by cryo-electron microscopy (cryo-EM) (Sherman et al. J Virol 80:10395-10406, 2006) but the structure of the RNA was only partially resolved in that study. To better understand the organization of the RNA and conformational changes resulting from the removal of divalent cations, small-angle neutron scattering with contrast variation experiments were performed. The results expand upon the cryo-EM results by clearly showing that virtually all of the RNA is contained in a thin shell that is in contact with the interior domains of the viral capsid protein, and they provide new insight into changes in the RNA packing that result from removal of divalent cations.

Mass spectrometric detection of targeting peptide bioconjugation to Red clover necrotic mosaic virus.

Plant virus nanoparticle (PVN) formulations constructed from Red clover necrotic mosaic virus by drug infusion and targeting peptide conjugation can be employed as drug delivery tools. In this investigation, we studied the cross-linked structures formed by application of sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) and succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester (SMPEG) as heterobifunctional linkers in the bioconjugation process. The plant virus formulations using several targeting peptides cross-linked to the plant virus capsid were characterized by LC/MS(E) analysis, which produced at least 69% sequence coverage using trypsin and chymotrypsin digestion. The results showed evidence for several types of modification located in three domains of the capsid protein. Extensive linker modifications on lysines or cysteines were detected in all the domains, including both intended peptide-capsid cross-links and unintended intracapsid cross-links. Surprisingly, the most extensive peptide modification was observed in the R domain, which is thought to be quite inaccessible to peptides and cross-linking reagents in solution, since it is on the interior of the virus. These results show that heterobifunctional linkers may not be the most efficient method for attachment of peptides to plant virus capsids. As an alternative conjugation strategy, maleimide peptides were used to conjugate with the virus in a one-step reaction. Analysis by LC/MS(E) showed that these one-step maleimide coupling reactions were more specific, such as modifications of C154 and to a lesser extent C267, and provide a means for achieving more effective PVN formulations.

Viruses as nanomaterials for drug delivery.

Virus delivery vectors are one among the many nanomaterials that are being developed as drug delivery materials. This chapter focuses on methods utilizing plant virus nanoparticles (PVNs) synthesized from the Red clover necrotic mosaic virus (RCNMV). A successful vector must be able to effectively carry and subsequently deliver a drug cargo to a specific target. In the case of the PVNs, we describe two types of ways cargo can be loaded within these structures: encapsidation and infusion. Several targeting approaches have been used for PVNs based on bioconjugate chemistry. Herein, examples of such approaches will be given that have been used for RCNMV as well as for other PVNs in the literature. Further, we describe characterization of PVNs, in vitro cell studies that can be used to test the efficacy of a targeting vector, and potential routes for animal administration.

Crystallization and preliminary X-ray diffraction analysis of red clover necrotic mosaic virus.

Red clover necrotic mosaic virus (RCNMV) is a species that belongs to the Tombusviridae family of plant viruses with a T = 3 icosahedral capsid. RCNMV virions were purified and were crystallized for X-ray analysis using the hanging-drop vapor-diffusion method. Self-rotation functions and systematic absences identified the space group as I23, with two virions in the unit cell. The crystals diffracted to better than 4 Šresolution but were very radiation-sensitive, causing rapid decay of the high-resolution reflections. The data were processed to 6 Šin the analysis presented here.

Infusion of dye molecules into Red clover necrotic mosaic virus.

The Red clover necrotic mosaic virus capsid is utilized to package and release molecules through reversible depletion and re-addition of divalent cations.

Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures.

Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.

The role of the C-terminal region of olive latent virus 1 coat protein in host systemic infection.

A full-length cDNA clone of olive latent virus 1 (OLV-1), a member of the genus Necrovirus, family Tombusviridae, was subjected to site-directed mutagenesis, and coat protein gene mutants were constructed. A mutant clone, denoted Delta3297, was obtained by deleting the nucleotide in position 3297, thus inducing a frameshift and replacing the last 49 amino acids of the viral coat protein (CP) by a shorter sequence of 39 amino acids. This mutant was viable, stable, able to synthesize a smaller CP, and able to give rise to the formation of apparently intact virus particles. Cell-to-cell movement of Delta3297 in Nicotiana benthamiana leaves was not affected, but, contrary to wild type OLV-1, it failed to spread systemically. These results indicate that virion formation is necessary but not sufficient for long-distance movement for OLV-1 and highlights the role of the CP carboxy-terminal domain in systemic infection.

A translational enhancer element on the 3'-proximal end of the Panicum mosaic virus genome.

Panicum mosaic virus (PMV) is a single-stranded positive-sense RNA virus in the family Tombusviridae. PMV genomic RNA (gRNA) and subgenomic RNA (sgRNA) are not capped or polyadenylated. We have determined that PMV uses a cap-independent mechanism of translation. A 116-nucleotide translational enhancer (TE) region on the 3'-untranslated region of both the gRNA and sgRNA has been identified. The TE is required for efficient translation of viral proteins in vitro. For mutants with a compromised TE, addition of cap analog, or transposition of the cis-active TE to another location, both restored translational competence of the 5'-proximal sgRNA genes in vitro.

Emergence of a resistance-breaking isolate of Rice yellow mottle virus during serial inoculations is due to a single substitution in the genome-linked viral protein VPg.

The recessive gene rymv-1, responsible for the high resistance of Oryza sativa 'Gigante' to Rice yellow mottle virus (genus Sobemovirus), was overcome by the variant CI4*, which emerged after serial inoculations of the non-resistance-breaking (nRB) isolate CI4. By comparison of the full-length sequences of CI4 and CI4*, a non-synonymous mutation was identified at position 1729, localized in the putative VPg domain, and an assay was developed based on this single-nucleotide polymorphism. The mutation G1729T was detected as early as the first passage in resistant plants and was found in all subsequent passages. Neither reversion nor any additional mutation was observed. The substitution G1729T, introduced by mutagenesis into the VPg of an nRB infectious clone, was sufficient to induce symptoms in uninoculated leaves of O. sativa 'Gigante'. This is the first evidence that VPg is a virulence factor in plants with recessive resistance against viruses outside the family Potyviridae.