We report the complete nucleotide sequences of lettuce infectious yellows virus (LIYV) RNAs 1 and 2. LIYV RNA 1 is 8118 nucleotides and includes three open reading frames (ORFs). Computer-assisted analysis of LIYV RNA 1 ORFs identified domains for a papain-like protease, methyltransferase (MTR), RNA helicase (HEL), and RNA-dependent RNA polymerase (RdRp). We suggest that the RdRp domain is expressed independently of the other replication-associated domains via a + 1 ribosomal frameshift. Amino acid sequences of the MTR, HEL, and RdRp show highly significant similarity to the homologous sequences from other closteroviruses and lower similarity to the respective proteins of tobamoviruses, tobraviruses, hordeiviruses, bromoviruses, and furoviruses. LIYV RNA 2 is 7193 nucleotides and includes six ORFs. These ORFs include a gene array that is characteristic of the closteroviruses: ORFs encoding a small membrane protein, a homologue of the HSP70 family of chaperone proteins, a protein whose function is unknown, the coat protein, and a diverged duplicate of the coat protein. LIYV is distinguished from the monopartite closteroviruses in the following ways: its genome consists of two RNAs, the positions of the coat protein gene and its diverged duplicate are reversed, and LIYV includes ORFs that are unrelated to ORFs found in other closteroviruses.

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Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived capped in vitro transcripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned cDNAs of a closterovirus genome.