Replication and recombination of the Red clover necrotic mosaic virus

Abstract

In this study, Red clover necrotic mosaic virus (RCNMV) was used to better understand the functions of replication proteins and to identify the terminal promoter element involved in viral replication. RCNMV genome contains two positive-sense, single-stranded RNAs. RNA-1 encodes two proteins essential for viral replication: p27 and p88. p88 is a fusion protein containing p27 at its N terminus and RNA dependent RNA polymerase motifs at its C-terminal domain. The function of p27 is not known. In this work, studies of RNA-1 chimerical clones between a highly infectious clone and a poorly infectious clone and subsequent mutagenesis demonstrated that the N-terminal 14 amino acids of p27 and p88 were required for efficient RNA replication. Sequence analysis indicated that it is possibly involved in membrane interaction. Another important aspect of viral replication is template recognition by the replicase at the 3' promoter. The 3' -29 nucleotides of both RCNMV RNA-1 and RNA-2 can be predicted to form an identical stem-loop structure (SLS). Mutational analysis of the SLS indicated that both the structure and the loop sequence were required for viral replication. Within the 5-nt loop region, three discontinuous nucleotides were identified as critical nucleotides for RNA-replicase interaction. The functional groups in these key nucleotides involved in replicase recognition are predicted. The 3' promoter element of RCNMV not only affects viral RNA replication but also influences transgenic recombination. RCNMV RNA-2 encodes a movement protein (MP) that is required for viral cell-to-cell movement and systemic infection. Transgenic Nicotiana benthamiana plants expressing different versions of MP mRNA neither resisted RCNMV nor complemented RNA-1 infection. However, systemic infection was observed in transgenic lines expressing 5' truncated MP mRNA when only RNA-1 was inoculated. Further analysis showed that the infection was resulted from nonhomologous RNA recombination events between infecting RNA-1 and MP transgene mRNA. A replicase-mediated template switch model of the transgenic recombination was proposed. The presence of the 3' promoter element in the transgene mRNA thus was a major factor determining transgenic recombination frequencies. As predicted from the model, transgene mRNA lacking the 3' promoter element would not be a good donor RNA for transgenic recombination. Consequently, no transgenic recombination was detected in transgenic plants expressing the 3' truncated MP mRNA upon inoculation with RCNMV RNA-1.