The RNA of maize chlorotic mottle virus - the essential virus in maize lethal necrosis disease - is translated via a panicum mosaic virus-like cap-independent translation element

Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), an emerging disease worldwide that often causes catastrophic yield loss. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that, like other tombusvirids, MCMV RNA contains a cap-independent translation element (CITE) in its 3’ untranslated region (UTR). The MCMV 3’ CITE (MTE) was mapped to nucleotides 4164-4333 in the genomic RNA. SHAPE probing revealed that the MTE is a 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 a m7GpppN cap structure, which is normally required for eIF4E to bind RNA. The MTE interaction with eIF4E suggests eIF4E may be a soft target for engineered resistance to MCMV. 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 the 3’-coterminal subgenomic RNA1 via long-distance kissing stem-loop base pairing to facilitate translation in wheat germ extract and in protoplasts. However, the MTE is a relatively weak stimulator of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs. 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. We propose that MCMV, and some other positive strand RNA viruses, favors a weak translation element to allow highly efficient viral RNA synthesis

Plant Virus Biodiversity and Ecology

The Plant Virus Biodiversity and Ecology (PVBE) project has been initiated to survey the biodiversity of viruses affecting vascular plants

Maize Chlorotic Mottle Machlomovirus and Wheat Streak Mosaic Rymovirus Concentrations Increase in the Synergistic Disease Corn Lethal Necrosis

AbstractCorn lethal necrosis (CLN) is caused by the synergistic interaction between maize chlorotic mottle machlomovirus (MCMV) and any potyvirus which infects cereals. Interactions between MCMV and wheat streak mosaic rymovirus (WSMV) in N28Ht corn produced MCMV concentrations that averaged 3.3- to 11.2-fold higher in doubly infected plants than the average concentrations in plants inoculated with MCMV. MCMV-negative sense RNA concentrations were similarly increased, and the ratio of full-length to subgenomic RNA was the same in singly and doubly infected plants. Contrary to most synergisms involving a potyvirus, WSMV infections were enhanced by the presence of MCMV. WSMV infection rates were higher when plants were coinoculated with MCMV, and the difference in infection rates was more pronounced at higher temperatures. Under conditions favorable for establishing high WSMV infection rates (cooler temperatures and high light intensity), WSMV concentrations in doubly infected plants averaged 2.1- to 3.1-fold higher than those in singly inoculated plants. Doubly inoculated plants with the lowest WSMV levels also had the lowest MCMV concentrations, but the concentrations of MCMV and WSMV in the most heavily infected plants did not directly correlate. These results suggest that there are genes in both MCMV and WSMV which directly or indirectly affect the replication and/or spread of the other virus in CLN

Maize chlorotic mottle virus.

Abstract In Peru, losses in floury and sweet maize varieties due to MCMV have been reported to average between 10 and 15%. In experimental plots, inoculated plant yields were reduced by up to 59% (Castillo, 1976). In Kansas crop losses due to corn lethal necrosis (caused by MCMV and any potyvirus) have been estimated to be between 50 (Uyemoto et al., 1980) and 90% (Niblett and Claflin, 1978) depending on the variety of maize and the year.</p

Maize Chlorotic Mottle Machlomovirus Expresses Its Coat Protein from a 1.47-kb Subgenomic RNA and Makes a 0.34-kb Subgenomic RNA

AbstractAnalysis of double-stranded RNAs produced in maize plants infected with maize chlorotic mottle machlomovirus (MCMV) and Northern blots of total RNA from infected plants or protoplasts showed two subgenomic RNAs (sgRNAs). Primer extension was used to map these sgRNAs, which are 1.47 and 0.34 kb long. The transcription start sites are nucleotide (nt) 2970 or 2971 for sgRNA1 and nt 4101 for sgRNA2. The 5′ ends of the sgRNAs are similar to one another and to the 5′ end of genomic RNA, and 11 nt sequences immediately upstream of their transcription start sites are similar. The location of the sgRNA1 transcription start site indicates that MCMV expresses a 7-kDa open reading frame (ORF) from nt 2995 to 3202 instead of the predicted 9-kDa ORF from nt 2959 to 3202. In protoplast inoculation experiments, a silent mutation at nt 2965 and a 4-nt change at nt 2959–2962 stopped the synthesis of sgRNA1 and expression of the coat protein ORF, which begins more than 400 nt downstream. Replication of MCMV does not require the expression of any of the ORFs encoded on sgRNA1. SgRNA2 has the potential to encode 2.3-, 2.7-, and 4.6-kDa peptides, but the function, if any, of sgRNA2 is unknown

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

AbstractMaize 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 RNA of maize chlorotic mottle virus - the essential virus in maize lethal necrosis disease - is translated via a panicum mosaic virus-like cap-independent translation element

AbstractMaize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), an emerging disease worldwide that often causes catastrophic yield loss. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that, like other tombusvirids, MCMV RNA contains a cap-independent translation element (CITE) in its 3’ untranslated region (UTR). The MCMV 3’ CITE (MTE) was mapped to nucleotides 4164-4333 in the genomic RNA. SHAPE probing revealed that the MTE is a 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 a m7GpppN cap structure, which is normally required for eIF4E to bind RNA. The MTE interaction with eIF4E suggests eIF4E may be a soft target for engineered resistance to MCMV. 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 the 3’-coterminal subgenomic RNA1 via long-distance kissing stem-loop base pairing to facilitate translation in wheat germ extract and in protoplasts. However, the MTE is a relatively weak stimulator of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs. 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. We propose that MCMV, and some other positive strand RNA viruses, favors a weak translation element to allow highly efficient viral RNA synthesis.Author SummaryIn recent years, maize lethal necrosis disease has caused massive crop losses in East Africa and Ecuador. It has also emerged in East Asia. 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 full immunity to MCMV. In order to design better resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression required for infection 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 in a way that differs from host mRNA interactions with the translational machinery. This difference may guide engineering of – or breeding for – resistance to MCMV. Moreover, this work adds to the diversity of known eukaryotic translation initiation mechanisms, as it provides more information on mRNA structural features that permit noncanonical interaction with a translation factor. Finally, owing to the conflict between ribosomes translating and viral replicase copying viral RNA, we propose that MCMV has evolved a relatively weak translation element in order to permit highly efficient RNA synthesis, and that this replication-translation trade-off may apply to other positive strand RNA viruses.</jats:sec