Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus

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.