The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-D(H)) was determined The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-D(H) cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with Δ22 and Δ82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p71, p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p71, p7a, p7b and CP are involved in TNV-D(H) cell-to-cell and longdistance movement, respectively.
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