The RNA of Maize Chlorotic Mottle Virus, an Obligatory Component of Maize Lethal Necrosis Disease, Is Translated via a Variant Panicum Mosaic Virus-Like Cap-Independent Translation Element.

Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), a serious emerging disease worldwide. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that MCMV RNA contains a cap-independent translation element (CITE) in its 3' untranslated region (UTR). The MCMV 3' CITE (MTE) was mapped to nucleotides 4164 to 4333 in the genomic RNA. 2'-Hydroxyl acylation analyzed by primer extension (SHAPE) probing revealed that the MTE is a distinct variant of the panicum mosaic virus-like 3' CITE (PTE). Like the PTE, electrophoretic mobility shift assays (EMSAs) indicated that eukaryotic translation initiation factor 4E (eIF4E) binds the MTE despite the absence of an m7GpppN cap structure, which is normally required for eIF4E to bind RNA. Using a luciferase reporter system, mutagenesis to disrupt and restore base pairing revealed that the MTE interacts with the 5' UTRs of both genomic RNA and subgenomic RNA1 via long-distance kissing stem-loop interaction to facilitate translation. The MTE stimulates a relatively low level of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs, mainly because the MTE lacks the pyrimidine-rich tract that base pairs to a G-rich bulge to form the pseudoknot. However, most mutations designed to form a pseudoknot decreased translation activity. Mutations in the viral genome that reduced or restored translation prevented and restored virus replication, respectively, in maize protoplasts and in plants. In summary, the MTE differs from the canonical PTE but falls into a structurally related class of 3' CITEs.IMPORTANCE In the past decade, maize lethal necrosis disease has caused massive crop losses in East Africa. It has also emerged in China and parts of South America. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer immunity to MCMV. In order to improve resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression by all positive-strand RNA viruses. We identified a structure (cap-independent translation element) in the 3' untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor, eIF4E, in a way that differs from host mRNA interactions with the translational machinery. This difference indicates eIF4E may be a soft target for engineering of-or breeding for-resistance to MCMV.

Tracing the Lineage of Two Traits Associated with the Coat Protein of the Tombusviridae: Silencing Suppression and HR Elicitation in Nicotiana Species.

In this paper we have characterized the lineage of two traits associated with the coat proteins (CPs) of the tombusvirids: Silencing suppression and HR elicitation in Nicotiana species. We considered that the tombusvirid CPs might collectively be considered an effector, with the CP of each CP-encoding species comprising a structural variant within the family. Thus, a phylogenetic analysis of the CP could provide insight into the evolution of a pathogen effector. The phylogeny of the CP of tombusvirids indicated that CP representatives of the family could be divided into four clades. In two separate clades the CP triggered a hypersensitive response (HR) in Nicotiana species of section Alatae but did not have silencing suppressor activity. In a third clade the CP had a silencing suppressor activity but did not have the capacity to trigger HR in Nicotiana species. In the fourth clade, the CP did not carry either function. Our analysis illustrates how structural changes that likely occurred in the CP effector of progenitors of the current genera led to either silencing suppressor activity, HR elicitation in select Nicotiana species, or neither trait.

Analysis of gene functions in Maize chlorotic mottle virus.

Gene functions of strains of Maize chlorotic mottle virus, which comprises the monotypic genus Machlomovirus, have not been previously identified. In this study mutagenesis of the seven genes encoded in maize chlorotic mottle virus (MCMV) showed that the genes with positional and sequence similarity to their homologs in viruses of related tombusvirid genera had similar functions. p50 and its readthrough protein p111 are the only proteins required for replication in maize protoplasts, and they function at a low level in trans. Two movement proteins, p7a and p7b, and coat protein, encoded on subgenomic RNA1, are required for cell-to-cell movement in maize, and p7a and p7b function in trans. A unique protein, p31, expressed as a readthrough extension of p7a, is required for efficient systemic infection. The 5' proximal MCMV gene encodes a unique 32kDa protein that is not required for replication or movement. Transcripts lacking p32 expression accumulate to about 1/3 the level of wild type transcripts in protoplasts and produce delayed, mild infections in maize plants. Additional studies on p32, p31 and the unique amino-terminal region of p50 are needed to further characterize the life cycle of this unique tombusvirid.

Pelarspovirus, a proposed new genus in the family Tombusviridae.

Currently, the family Tombusviridae encompasses thirteen viral genera that contain single-stranded, positive-sense RNA genomes and isometric virions; the exception being the genus Umbravirus, whose members do not encode a coat protein (CP). A new genus, tentatively named Pelarspovirus, is proposed to be added to this family and would include five members, with Pelargonium line pattern virus recommended as the type species. Viruses assigned to this proposed genus have monopartite genomes encoding five open reading frames (ORFs) that include two 5'-proximal replication proteins, two centrally located movement proteins (MP1 and MP2) and a 3'-proximal CP that, at least for pelargonium line pattern virus (PLPV), has been shown to act also as suppressor of RNA silencing. Distinguishing characteristics of these viruses include i) production of a single, tricistronic subgenomic RNA for expression of MP and CP genes, ii) presence of a non-AUG start codon (CUG or GUG) initiating the MP2 ORF, iii) absence of AUG codons in any frame between the AUG initiation codons of MP1 and CP genes, and iv) sequence-based phylogenetic clustering of all encoded proteins in separate clades from those of other family members.

Infectious transcripts of an asymptomatic panicovirus identified from a metagenomic survey.

A new panicovirus was identified from a metagenomic survey of plant viruses in the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, USA. Thin paspalum asymptomatic virus (TPAV) was isolated from Paspalum setaceum var. muehlenbergii. The complete 4195 nucleotide sequence encodes six proteins of 44, 109, 8, 6.8, 26, and 15kDa, whose genes are similar in size and location to those of panicum mosaic virus (PMV) and cocksfoot mild mosaic virus (CMMV). Infectious transcripts made from cDNAs of the 2005 isolate were used for initial host range analyses. Of the 11 tested grasses, only Paspalum setaceum (var. stramineum), Setaria italica, and Setaria viridis were hosts, producing asymptomatic infections, and Nicotiana benthamiana was not infected. Protein and RNA alignments indicate that TPAV is more closely related to PMV than CMMV. RNA alignments and structure predictions indicate that TPAV and CMMV have conserved eight structural elements previously predicted or analyzed for PMV. The initial TPP specimen also contained RNAs related to alphacryptoviruses. Partial sequences of RNA1 (∼35%) and RNA2 (∼40%) of putative thin paspalum cryptic virus were obtained. These represent the first sequences for a putative grass-infecting alphacryptovirus.

Detection of members of the Tombusviridae in the Tallgrass Prairie Preserve, Osage County, Oklahoma, USA.

Viruses are most frequently discovered because they cause disease in organisms of importance to humans. To expand knowledge of plant-associated viruses beyond these narrow constraints, non-cultivated plants of the Tallgrass Prairie Preserve, Osage County, Oklahoma, USA were systematically surveyed for evidence of the presence of viruses. This report discusses viruses of the family Tombusviridae putatively identified by the survey. Evidence of two carmoviruses, a tombusvirus, a panicovirus and an unclassifiable tombusvirid was found. The complete genome sequence was obtained for putative TGP carmovirus 1 from the legume Lespedeza procumbens, and the virus was detected in several other plant species including the fern Pellaea atropurpurea. Phylogenetic analysis of the sequence and partial sequence of a related virus supported strongly the placement of these viruses in the genus Carmovirus. Polymorphisms in the sequences suggested existence of two populations of TGP carmovirus 1 in the study area and year-to-year variations in infection by TGP carmovirus 3.

The cap-binding translation initiation factor, eIF4E, binds a pseudoknot in a viral cap-independent translation element.

Eukaryotic initiation factor eIF4E performs a key early step in translation by specifically recognizing the m7GpppN cap structure at the 5' end of cellular mRNAs. Many viral mRNAs lack a 5' cap and thus bypass eIF4E. In contrast, we reported a cap-independent translation element (PTE) in Pea enation mosaic virus RNA2 that binds and requires eIF4E for translation initiation. To understand how this uncapped RNA is bound tightly by eIF4E, we employ SHAPE probing, phylogenetic comparisons with new PTEs discovered in panico- and carmoviruses, footprinting of the eIF4E binding site, and 3D RNA modeling using NAST, MC-Fold, and MC-Sym to predict a compact, 3D structure of the RNA. We propose that the cap-binding pocket of eIF4E clamps around a pseudoknot, placing a highly SHAPE-reactive guanosine in the pocket in place of the normal m7GpppN cap. This reveals a new mechanism of mRNA recognition by eIF4E.

Infectious cDNA transcripts of Maize necrotic streak virus: infectivity and translational characteristics.

Maize necrotic streak virus (MNeSV) is a unique member of the family Tombusviridae that is not infectious by leaf rub inoculation and has a coat protein lacking the protruding domain of aureusviruses, carmoviruses, and tombusviruses (Louie et al., Plant Dis. 84, 1133-1139, 2000). Completion of the MNeSV sequence indicated a genome of 4094 nt. RNA blot and primer extension analysis identified subgenomic RNAs of 1607 and 781 nt. RNA and protein sequence comparisons and RNA secondary structure predictions support the classification of MNeSV as the first monocot-infecting tombusvirus, the smallest tombusvirus yet reported. Uncapped transcripts from cDNAs were infectious in maize (Zea mays L.) protoplasts and plants. Translation of genomic and subgenomic RNA transcripts in wheat germ extracts indicated that MNeSV has a 3' cap-independent translational enhancer (3'CITE) located within the 3' 156 nt. The sequence, predicted structure, and the ability to function in vitro differentiate the MNeSV 3'CITE from that of Tomato bushy stunt virus.

The dominant inhibitory chalcone synthase allele C2-Idf (inhibitor diffuse) from Zea mays (L.) acts via an endogenous RNA silencing mechanism.

The flavonoid pigment pathway in plants has been used as a model system for studying gene regulatory mechanisms. C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in this biosynthetic pathway of maize. Homozygous C2-Idf plants show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. To explore the nature of this effect, the C2-Idf allele was cloned. The gene structure of the C2-Idf haplotype differs substantially from that of the normal c2 gene in that three copies are present. Two of these are located in close proximity to each other in a head-to-head orientation and the third is closely linked. Previous experiments showed that the lower level of pigmentation in heterozygotes is correlated with reduced enzyme activity and low steady-state mRNA levels. We found that c2 transcription occurs in nuclei of C2-Idf/C2 heterozygotes, but mRNA does not accumulate, suggesting that the inhibition is mediated by RNA silencing. Infection of C2-Idf/C2 heterozygotes with viruses that carry suppressors of RNA silencing relieved the phenotypic inhibition, restoring pigment production and mRNA levels. Finally, we detected small interfering RNAs (siRNAs) in plants carrying C2-Idf, but not in plants homozygous for the wild-type C2 allele. Together, our results indicate that the inhibitory effect of C2-Idf occurs through RNA silencing.