Resistance of melon (Cucumis melo L.) to Melon necrotic spot virus (MNSV) is inherited as a single recessive gene, denoted nsv. No MNSV isolates described to date (e.g., MNSV-Malpha5), except for the MNSV-264 strain described here, are able to overcome the resistance conferred by nsv. Analysis of protoplasts of susceptible (Nsv/-) and resistant (nsv/nsv) melon cultivars inoculated with MNSV-264 or MNSV-Malpha5 indicated that the resistance trait conferred by this gene is expressed at the single-cell level. The nucleotide sequence of the MNSV-264 genome has a high nucleotide identity with the sequences of other MNSV isolates, with the exception of its genomic 3'-untranslated region (3'-UTR), where less than 50% of the nucleotides are shared between MNSV-264 and the other two MNSV isolates completely sequenced to date. Uncapped RNAs transcribed from a full-length MNSV-264 cDNA clone were infectious and caused symptoms indistinguishable from those caused by the parental viral RNA. This cDNA clone allowed generation of chimeric mutants between MNSV-264 and MNSV-Malpha5 through the exchange of the last 74 nucleotides of their coat protein (CP) open reading frames and the complete 3'-UTRs. Analysis of protoplasts of susceptible and resistant melon cultivars inoculated with chimeric mutants clearly showed that the MNSV avirulence determinant resides in the exchanged region. The carboxy-termini of the CP of both isolates are identical; therefore, the avirulence determinant likely consists of the RNA sequence itself. We also demonstrated that this genomic region contains the determinant for the unique ability of the isolate MNSV-264 to infect noncucurbit hosts (Nicotiana benthamiana and Gomphrena globosa).