Spontaneous deletion enhances movement of a cucumber necrosis virus based chimera expressing the red clover necrotic mosaic virus movement protein gene†

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Abstract
The 35‐kDa movement protein (MP) gene of red clover necrotic mosaic virus (RCNMV) and 3′ flanking sequence were inserted in a cucumber necrosis virus (CNV) deletion mutant lacking a large portion of the coding region for the MP. Nicotiana benthamiana plants inoculated with chimeric synthetic transcripts of the resulting hybrid cDNA clone (M5/RM2) developed both local and systemic symptoms and accumulated high levels of chimeric viral RNA. Reverse transcriptase polymerase chain reaction (RT‐PCR) and sequence analysis of viral RNA extracted from systemically infected leaves of four different plants revealed that in each plant a large portion (305, 308, 315 or 127 nts) of the 3′ terminus of the inserted sequence spontaneously deleted during infection. In three of the deletion derivatives, the truncated RCNMV MP open reading frame (ORF) was fused in‐frame with the remaining portion of the 3′ terminal region of CNV MP ORF. The movement efficiencies of M5/RM2, a cloned copy of one of the deletion derivatives (ClM5/RM2dd1), and a stop codon mutant of ClM5/RM2dd1 (ClM5/RM2dd1stop), which prevents translational fusion to the CNV MP, were compared and it was determined that deletion of RCNMV MP sequences in conjunction with fusion to CNV MP sequences increases the movement efficiency of the chimeric virus genome. Absence of the C‐terminal region of the RCNMV MP in RCNMV RNA‐2 abolished RCNMV movement. However, movement could be complemented in trans if cells were coinoculated with ClM5/RM2dd1. Complementation of RCNMV movement did not occur using ClM5/RM2dd1stop, suggesting a role for appended CNV MP sequences in movement of the RCNMV genome. The ability of the CNV replicase to delete unnecessary or deleterious RCNMV sequences and to append the required CNV MP sequences reinforces the role of RNA recombination in the adaptation and evolution of viral genomes.