Uncoating Mechanism of Carnation Mottle Virus Revealed by Cryo-EM Single Particle Analysis.

Genome uncoating is a prerequisite for the successful infection of plant viruses in host plants. Thus far, little is known about the genome uncoating of the Carnation mottle virus (CarMV). Here, we obtained two reconstructions of CarMV at pH7 in the presence (Ca-pH7) and absence (EDTA-pH7) of calcium ions by Cryo-EM single particle analysis, which achieved 6.4 Å and 8 Å resolutions respectively. Our results showed that chelation of the calcium ions under EDTA-pH7 resulted in reduced interaction between the subunits near the center of the asymmetric unit but not overall size change of the viral particles, which indicated that the role of the calcium ions in CarMV was not predominantly for the structural preservation. Part of the genomic RNA closest to the capsid was found to be located near the center of the asymmetric unit, which might result from the interaction between genomic RNA and Lys194 residues. Together with the electrostatic potential analysis on the inner surface of the asymmetric unit, the reduced interaction near the center of the asymmetric unit under EDTA-pH7 suggested that the genome release of CarMV might be realized through the center of the asymmetric unit.

A spectrum of HRT-dependent hypersensitive responses elicited by the 52 amino acid N-terminus of turnip crinkle virus capsid protein and its mutants.

The capsid protein (CP) of turnip crinkle virus (TCV) is the elicitor of hypersensitive response (HR) and resistance mediated by the resistance protein HRT in the Di-17 ecotype of Arabidopsis. Here we identified the N-terminal 52-amino-acid R domain of TCV CP as the elicitor of HRT-dependent HR in Nicotiana benthamiana. Mutating this domain at position 6 (R6A), but not at positions 8 (R8A) or 14 (G14A), abolished HR in N. benthamiana. However, on Di-17 Arabidopsis leaves only R8A R domain elicited visible epidermal HR. When incorporated in infectious TCV RNAs, R8A and G14A mutations exerted dramatically different effects in Di-17 plants, as R8A caused systemic cell death whereas G14A led to complete restriction of the mutant virus. This continual spectrum of HR and resistance responses elicited by various R domain mutants suggests that the CP-HRT interaction could be perturbed by conformational changes in the R domain of TCV CP.

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.

Membrane insertion and biogenesis of the Turnip crinkle virus p9 movement protein.

Plant viral infection and spread depends on the successful introduction of a virus into a cell of a compatible host, followed by replication and cell-to-cell transport. The movement proteins (MPs) p8 and p9 of Turnip crinkle virus are required for cell-to-cell movement of the virus. We have examined the membrane association of p9 and found that it is an integral membrane protein with a defined topology in the endoplasmic reticulum (ER) membrane. Furthermore, we have used a site-specific photo-cross-linking strategy to study the membrane integration of the protein at the initial stages of its biosynthetic process. This process is cotranslational and proceeds through the signal recognition particle and the translocon complex.

Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3' UTR of turnip crinkle virus.

The 3(') untranslated region (3(') UTR) of turnip crinkle virus (TCV) genomic RNA contains a cap-independent translation element (CITE), which includes a ribosome-binding structural element (RBSE) that participates in recruitment of the large ribosomal subunit. In addition, a large symmetric loop in the RBSE plays a key role in coordinating the incompatible processes of viral translation and replication, which require enzyme progression in opposite directions on the viral template. To understand the structural basis for the large ribosomal subunit recruitment and the intricate interplay among different parts of the molecule, we determined the global structure of the 102-nt RBSE RNA using solution NMR and small-angle x-ray scattering. This RNA has many structural features that resemble those of a tRNA in solution. The hairpins H1 and H2, linked by a 7-nucleotide linker, form the upper part of RBSE and hairpin H3 is relatively independent from the rest of the structure and is accessible to interactions. This global structure provides insights into the three-dimensional layout for ribosome binding, which may serve as a structural basis for its involvement in recruitment of the large ribosomal subunit and the switch between viral translation and replication. The experimentally determined three-dimensional structure of a functional element in the 3(') UTR of an RNA from any organism has not been previously reported. The RBSE structure represents a prototype structure of a new class of RNA structural elements involved in viral translation/replication processes.

The 3' end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase.

Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 3' translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 3' end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 3' end, which causes structural changes that potentiate the shift between translation and replication.

Preliminary X-ray data analysis of crystalline hibiscus chlorotic ringspot virus.

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3.2 A resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 A. Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV.

Characterization of the subgenomic RNAs produced by Pelargonium flower break virus: Identification of two novel RNAs species.

Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, has a single-stranded positive-sense genomic RNA (gRNA) of 3.9kb. The 5' half of the gRNA encodes two proteins involved in replication, the p27 and its readthrough product, p86 (the viral RNA dependent-RNA polymerase, RdRp), and the 3' half encodes two small movement proteins, p7 and p12, and the coat protein (CP). As other members of the family Tombusviridae, carmoviruses express ORFs that are not 5'-proximal from subgenomic RNAs (sgRNAs). Analysis of double-stranded RNAs extracted from PFBV-infected leaves and Northern blot hybridizations of total RNA from infected plants or protoplasts revealed than PFBV produces four 3'-coterminal sgRNAs of 3.2, 2.9, 1.7 and 1.4kb, respectively. The 5' termini of the 1.7 and 1.4kb sgRNAs mapped 26 and 143 nt upstream of the initiation codons of the p7 and CP genes, respectively, whereas the 5'-ends of the 3.2 and 2.9kb sgRNAs were located within the readthrough portion of the RdRp gene. The PFBV sgRNAs begin with a motif which is also present at the 5' terminus of the gRNA and the minus polarity of the regions preceding their corresponding start sites (in the gRNA) may be folded into hairpin structures resembling those found for the sgRNA promoters of other carmoviruses. The results indicate that, besides the sgRNAs involved in the translation of the movement proteins and the CP identified in most carmoviral infections, PFBV produces two additional sgRNAs whose biological significance is currently unknown. The possible participation of the 3.2 and 2.9kb PFBV sgRNAs in the expression of readthrough portions of the RdRp is discussed.

Structural domains within the 3' untranslated region of Turnip crinkle virus.

The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3' end of Turnip crinkle virus (TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (Psi(3)) within the 3'-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, Psi(3) and a second pseudoknot (Psi(2)) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, Psi(3) and a pseudoknot that connects H5 and the 3' end (Psi(1)). Stepwise replacements of TCV elements with the comparable elements from Cardamine chlorotic fleck virus indicated that the complete 142-nt 3' end, and subsets containing Psi(3), H4a, and H4b or Psi(3), H4a, H4b, H5, and Psi(2), form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, Psi(3), and Psi(2) are capable of simultaneous existence and bears some resemblance to a tRNA. The related Japanese iris necrotic ring virus does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3' untranslated region sequences such as translation and replication.

Structure of Cowpea mottle virus: a consensus in the genus Carmovirus.

Cowpea mottle virus (CPMoV) is a T = 3 virus that belongs to Carmovirus genus of the Tombusviridae family. Here, we report the crystal structure of CPMoV determined to a resolution of 7.0 angstroms. The structures and sequences of three Carmoviruses, CPMoV, Turnip crinkle virus (TCV), and Carnation mottle virus (CarMV) have been compared to TBSV from the Tombusvirus genus. CPMoV, TCV, and CarMV all have a deletion in betaC strand in the S domain relative to TBSV that may be distinctive to the genus. Although CPMoV has an elongated C-terminus like TBSV, it does not interact with the icosahedrally related P domain as observed in TBSV. In CPMoV, the termini of A and B interact with the icosahedrally related shell domains of A and C, respectively, to form a chain of interactions around the 5-fold axes. The C subunit terminus does not, however, interact with the B subunit because of quasi-equivalent differences in the P domain orientations.

Three-dimensional reconstruction of hibiscus chlorotic ringspot virus.

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense, single-stranded RNA virus, which belongs to the Tombusviridae family and infects plants of the Hibiscus genus, including kenaf, a woody plant of agricultural importance. These icosahedral viruses have a capsid consisting of 180 copies of coat protein (CP) arranged with T=3 symmetry. The CP consists of an internal RNA-binding domain, a shell-forming domain and a protruding domain. The HCRSV virion was reconstructed to about 12A resolution from cryo-EM images using the program EMAN. The structure had the arrangement of 90 dimers of protruding domains characteristic of the Tombusviridae. Reconstructions were also made from negatively stained samples, and showed essentially the same features. In addition, a particle of a different, "smooth" appearance was also identified in the negatively stained samples. These particles were slightly smaller and lacked protruding domains. Biochemical analysis confirmed the presence of two protein products: a 37 kDa protein identified as HCRSV CP and a 54 kDa protein that appeared to be of non-HCRSV origin.

Purification, crystallization and X-ray analysis of Hibiscus chlorotic ringspot virus.

Hibiscus chlorotic ringspot virus (HCRSV), a Carmovirus, occurs worldwide and induces chlorotic ringspots on leaves, stunting and flower distortion in Hibiscus species, including kenaf. The HCRSV capsid has T = 3 icosahedral symmetry and contains 180 copies of the coat protein. A virus yield of 48-70 mg per 100 g of infected kenaf leaves was achieved with an improved purification scheme involving sucrose-cushion and sucrose density-gradient centrifugation. The virus was crystallized using PEG 8000 and 2,3-butanediol as co-precipitants. The crystals belonged to the cubic space group P23, with unit-cell parameter a = 392 A, and diffracted X-rays to at least 4.5 A resolution.

Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants.

Time-course and in situ hybridization analyses were used to study the spatio-temporal distribution of Carnation mottle virus (CarMV) in Chenopodium quinoa plants. Genomic and subgenomic RNAs of plus polarity accumulated linearly with time, whereas the corresponding minus strands reached a peak during infection in inoculated leaves. Analyses of serial tissue sections showed that plus polarity strands were localized throughout the infection area, whereas minus strands were localized at the borders of the chlorotic lesions. The accumulation kinetics of the coat protein (CP) and the p7 movement protein (MP) as well as their subcellular localization were also studied. Unlike most MPs, CarMV p7 showed a non-transient expression and a mainly cytosolic location. However, as infection progressed the presence of p7 in the cell wall fraction increased significantly. These results are discussed on the basis of a recent model proposed for the mechanism of cell-to-cell movement operating in the genus Carmovirus.

Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane.

Virus-encoded movement proteins (MPs) mediate cell-to-cell spread of viral RNA through plant membranous intercellular connections, the plasmodesmata. The molecular pathway by which MPs interact with viral genomes and target plasmodesmata channels is largely unknown. The 9-kDa MP from carnation mottle carmovirus (CarMV) contains two potential transmembrane domains. To explore the possibility that this protein is in fact an intrinsic membrane protein, we have investigated its insertion into the endoplasmic reticulum membrane. By using in vitro translation in the presence of dog pancreas microsomes, we demonstrate that CarMV p9 inserts into the endoplasmic reticulum without the aid of any additional viral or plant host components. We further show that the membrane topology of CarMV p9 is N(cyt)-C(cyt) (N and C termini of the protein facing the cytoplasm) by in vitro translation of a series of truncated and full-length constructs with engineered glycosylation sites. Based on these results, we propose a topological model in which CarMV p9 is anchored in the membrane with its N- and C-terminal tail segments interacting with its soluble, RNA-bound partner CarMV p7, to accomplish the viral cell-to-cell movement function.

Comparative study on genomes of two Japanese melon necrotic spot virus isolates.

Nucleotide sequences of the genomes of two Japanese Melon necrotic spot virus (MNSV) isolates, NH and NK were determined. The open reading frames (ORFs) in both genomes encode five proteins: p29 (the pre-readthrough domain of p89), p89 (the readthrough domain of p89 identified as the putative RNA-dependent RNA polymerase), p14 (the pre-readthrough domain of p7A), p7A (the putative movement protein), and p42 (coat protein, CP). Nucleotide and amino acid sequence identities of the five proteins of NH and NK isolates were estimated at 97.4-99.5% and 97.7-100%, respectively. NK isolate but not NH isolate infected systemically leaves of Cucumis melo plants. When deduced amino acid sequences of p7A proteins of NH and NK isolates were compared, only one difference at position 16 (serine in NH isolate and isoleucine in NK isolate) was observed. p7A protein is considered the putative movement protein. The serine of p7A protein of NH isolates may be involved in systemic infection. In addition, phylogenetic relationships of genes based on nucleotide sequences revealed that NH and NK isolates might form a group, and S isolate, serologically different from NH and NK isolates, might represent a distinct isolate not belonging to this group.