Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease.

The sequence of 8734 nucleotides (nt) from the 5'-end of the beet yellows closterovirus (BYV) RNA was determined to complete the 15,480-nt sequence of the virus genome. The 5'-terminal two-thirds of the sequence are occupied by two overlapping open reading frames (ORFs) 1a and 1b, encoding products with calculated M(r) of 295K and 48K, respectively. The RNA sequence surrounding the stop codon in ORF 1a shows structural elements typical of ribosomal frameshifting signals in a number of animal and plant viruses. It is predicted that the ORF 1b product is expressed via a +1 ribosomal frameshifting as the 348K ORF 1a/1b fusion protein. This putative protein contains the array of methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains that is conserved in the Sindbis-like supergroup of positive-strand RNA viruses. The 348K protein of BYV is longer than the putative replicases of the most closely related viruses (tobra- and tobamoviruses) by about 1300 amino acids distributed between two unique regions, one at the N-terminus, and the other in the central portion. The N-terminal domain showed sequence similarity to the helper component papain-like protease of potyviruses. By using in vitro translation of the T7 transcripts encoding the N-terminal 92K peptide of the BYV ORF 1a product, we found that the N-terminal fragment of 588 amino acids is released from the translation product by cleavage at the Gly-Gly dipeptide. Site-directed mutagenesis of either of the predicted catalytic residues Cys-509 and His-569 or of the Gly-588 at the cleavage site completely abolished the cleavage. The central unique region of the 348K protein contains a domain distantly resembling the aspartic protease of HIV and other lentiviruses. As shown previously, the 3'-terminal portion of the BYV genome encompasses seven more ORFs, one of which codes for a protein related to the HSP70 cell heat shock proteins, whereas two others encode the capsid protein and its diverged copy. Thus, despite the apparent evolutionary relationship with Sindbis-like viruses, BYV comprises a collection of genomic modules absorbed from different sources and has a unique expression strategy.