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Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot
J A Díaz1, J J Bernal, E Moriones
1Estación Experimental La Mayora, Consejo Superior de Investigaciones Científicas, Málaga, Spain.
Abstract:
We have studied the biological and molecular characteristics of a MNSV isolate collected in Spain (MNSV-Malpha5) and generated a full-length cDNA clone from which infectious RNA transcripts can be produced. The host range of MNSV-Malpha5 appeared to be limited to cucurbits and did not differ from that of MNSV-Dutch [4, 21]. However, differences were observed in the type of symptoms that both isolates could induce. A full-length cDNA of MNSV-Malpha5 was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a 5'-end primer anchoring a T7 RNA promoter sequence and a 3'-end primer, and cloned. Uncapped RNAs transcribed from this cDNA clone were infectious and caused symptoms indistinguishable from those caused by viral RNA when mechanically inoculated onto melon, cucumber or watermelon plants. The complete genome sequence of MNSV-Malpha5 was deduced from the full length cDNA clone. It is 4271 nt long and, similarly to MNSV-Dutch, consists of 5' and 3' untranslated regions (UTRs) and five open reading frames (ORFs) coding for 29, 89, 42 and two small 7 kDa proteins. One notable difference between MNSV-Malpha5 and other sequenced MNSV isolates was found, as for MNSV-Malpha5 the first of the two small ORFs, which are contiguous in the genome, terminates with a genuine stop codon, whereas for MNSV-Dutch and other sequenced MNSV isolates it terminates with an amber codon. This suggested that the putative p14 readthrough protein that could be expressed from the MNSV-Dutch and other MNSV genomes could not be expressed from the MNSV-Malpha5 genome. Also, the nucleotide and amino acid sequences comparisons showed a distant relationship of MNSV-Malpha5 with other known MNSV isolates.
Insights
This study characterizes a Spanish Melon Necrotic Spot Virus (MNSV) isolate, MNSV-Malpha5. Researchers created a cDNA clone, confirming its infectiousness and revealing unique genetic differences compared to other MNSV strains.
Area of Science:
- Plant Virology
- Molecular Biology
- Genomics
Background:
- Melon Necrotic Spot Virus (MNSV) is a significant pathogen affecting cucurbits.
- Understanding MNSV isolate diversity is crucial for disease management.
Purpose of the Study:
- To biologically and molecularly characterize a Spanish MNSV isolate (MNSV-Malpha5).
- To generate a full-length cDNA clone for producing infectious RNA transcripts.
- To compare MNSV-Malpha5 with other known MNSV isolates.
Main Methods:
- Generation of a full-length cDNA clone of MNSV-Malpha5.
- Reverse-transcription polymerase chain reaction (RT-PCR) for cDNA amplification.
- Mechanical inoculation of cucurbit plants (melon, cucumber, watermelon) with transcribed RNA.
- Genome sequencing and comparative analysis of nucleotide and amino acid sequences.
Main Results:
- MNSV-Malpha5 exhibits a host range limited to cucurbits, similar to MNSV-Dutch, but induces distinct symptoms.
- Infectious RNA transcripts were successfully produced from the cDNA clone, causing characteristic MNSV symptoms.
- The complete genome sequence of MNSV-Malpha5 is 4271 nucleotides long, with five open reading frames (ORFs).
- A key genetic difference was identified: MNSV-Malpha5 has a genuine stop codon in a small ORF, unlike the amber codon in other MNSV isolates, potentially preventing p14 readthrough protein expression.
- Sequence comparisons indicated a distant relationship between MNSV-Malpha5 and other MNSV isolates.
Conclusions:
- The generated MNSV-Malpha5 cDNA clone is a valuable tool for studying MNSV biology and pathogenesis.
- MNSV-Malpha5 represents a distinct genetic lineage within the MNSV species.
- The identified genetic variation may influence viral protein expression and host-pathogen interactions.