To study the proteolytic processing of the potato leafroll virus replicase proteins, the multidomain P1 protein with a c-myc epitope tag attached at the N terminus was expressed in insect cells by using the baculovirus system. Western blotting showed that P1 was cleaved at a site upstream of the serine protease domain, in addition to the cleavage site downstream of the protease domain. Mutational analysis showed that the serine protease domain within P1 was responsible for this cleavage. To characterize this novel cleavage site further, a portion of the P1 protein comprising the protease domain and the two cleavage sites was expressed in Escherichia coli. A similar cleavage event was observed in bacteria and was abolished when the P1 protease was inactivated by mutation. Peptide-sequencing studies indicated that this cleavage occurred at a Glu/Arg junction, separating the N-terminal 204 residues from the serine protease domain of P1.Potato leafroll virus (PLRV) is now classified as the type member of the genus Polerovirus of the family Luteoviridae, which also contains the genera Luteovirus and Enamovirus (Pringle, 1998). PLRV virions appear to be non-enveloped, isometric particles with a diameter of 24 nm (Harrison, 1984). Viral particles contain a single-stranded (ss), positive-sense genomic RNA of some 5.8 kb in length (Mayo et al., 1989;van der Wilk et al., 1989;Keese et al., 1990). Like most luteoviruses, PLRV genomic RNA encodes six major open reading frames (ORFs), which are arranged into two blocks separated by a small intergenic region. ORF0 encodes a 28 kDa protein that is important for virus accumulation (Sadowy et al., 2001b). Site-directed mutagenetic analysis has shown that P1 and P2, encoded by ORF1 and ORF2, are required for virus replication (Sadowy et al., 2001a) -consistent with studies on beet western yellows virus (BWYV), another polerovirus (Reutenauer et al., 1993). The 39-terminal block encodes the capsid protein (ORF3), the putative movement protein (ORF4) and the aphid transmission protein (ORF5; reviewed by Miller et al., 1995;Mayo & Ziegler-Graff, 1996). In addition to these six ORFs, two small ORFs were identified within the 39-terminal region (Ashoub et al., 1998). Jaag et al. (2003) reported that another small ORF within the P1 region, in an alternative reading frame, was essential for viral multiplication.Like many positive-strand ssRNA viruses, PLRV has been found to express the products of its genome in a variety of ways, including transcription of subgenomic mRNAs, translational frameshifting, stop-codon readthrough, internal initiation, translation initiation at an internal ribosomal entry site and polyprotein processing (Miller et al., 1995;Mayo & Ziegler-Graff, 1996).Proteins encoded within the 59-proximal region are translated from the genomic RNA, whereas 39-proximal ORFs are expressed from two subgenomic RNAs (Tacke et al., 1990;Miller & Mayo, 1991;Ashoub et al., 1998). ORF2, which encodes the RNA-dependent RNA polymerase, is expressed via 21 frameshifting at a site withi...