The entire genome of grapevine leafroll-associated closterovirus-2 (GLRaV-2), except the exact 5' terminus, was cloned and sequenced. The sequence encompasses nine open reading frames (ORFs) which include, in the 5' to 3' direction, an incomplete ORF1a encoding a putative viral polyprotein and eight ORFs that encode proteins of 52 kDa (ORF1b), 6 kDa (ORF2), 65 kDa (ORF3), 63 kDa (ORF4), 25 kDa (ORF5), 22 kDa (ORF6), 19 kDa (ORF7) and 24 kDa (ORF8) respectively, and 216 nucleotides of the 3' untranslated region. An incomplete ORF1a potentially encoded a large polyprotein containing the conserved domains characteristic of a papain-like protease, methyltransferase and helicase. ORF1b potentially encoded a putative RNA-dependent RNA polymerase. The expression of ORF1b may be via a +1 ribosomal frameshift mechanism, similar to other closteroviruses. A unique gene array, which is conserved in other closteroviruses, was also identified in GLRaV-2; it includes genes encoding a 6 kDa small hydrophobic protein, 65 kDa heat shock protein 70, 63 kDa protein of function unknown, 25 kDa coat protein duplicate and 22 kDa coat protein. Identification of ORF6 (22 kDa) as the coat protein gene was further confirmed by in vivo expression in E. coli and immunoblotting. Phylogenetic analysis comparing different genes of GLRaV-2 with those of other closteroviruses demonstrated a close relationship with beet yellows virus (BYV), beet yellow stunt virus and citrus tristeza virus. GLRaV-2 is the only closterovirus, so far, that matches the genome organization of the type member of the group, BYV, and thus can be unambiguously classified as a definitive member of the genus Closterovirus.


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