Identification of genes involved in replication and movement of peanut clump virus

The genome of peanut clump pecluvirus (PCV) consists of two messenger RNA components which contain, respectively, three and five open reading frames (ORFs). Inoculation of transcripts from full-length cDNA clones derived from the PCV RNAs showed that RNA-1 is able to replicate in the absence of RNA-2 in protoplasts, but both RNAs are necessary for plant infection. To investigate the role of different gene products in viral RNA replication and movement, transcripts from mutant cDNA clones were inoculated to protoplasts and to Chenopodium quinoa or Nicotiana benthamiana plants, and progeny RNA was detected by Northern blot analysis. The protein P15, encoded by the third ORF of RNA-1, is essential for efficient replication of the viral genome. The three proteins, P51, P14, and P17, of the triple gene block contained in RNA-2 are involved in localized movement of the viral genome, whereas the coat protein (P23) is also required for vascular movement. Insertion of the beta-glucuronidase reporter gene (GUS) in place of the P23 or P39 genes (the first and the second genes of RNA-2) allows visualization of the virus infection in inoculated leaves. Although the presence of the GUS gene resulted in a lower accumulation of progeny RNA and, despite instability of the construct in planta, histochemical detection of PCV multiplication was more sensitive than Northern blot detection.