We present fine mapping of a cis-acting nucleotide sequence found in the 5 region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3 region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5 region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquitoborne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.Flaviviruses are small enveloped viruses. The virion contains a positive-strand RNA genome of about 10.7 kb encapsidated by 180 copies of the capsid (C) protein in a nucleocapsid core, which is enveloped by a lipid bilayer in which 180 copies of each of two transmembrane proteins, envelope (E) and membrane (M), are anchored (2, 9). The flaviviruses include a large number of serious human pathogens such as yellow fever virus (YFV), four serotypes of dengue virus (DENV), Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus. Although good vaccines have been developed against some flaviviruses (YFV, Japanese encephalitis virus, and tickborne encephalitis virus), they remain a serious health risk around the world.The flavivirus RNA contains one large open reading frame flanked by 5Ј and 3Ј nontranslated regions that are required for replication and translation of the RNA (11). Analysis of the sequences of several flavivirus RNAs showed that short sequences close to the 5Ј and 3Ј ends are complementary, and we proposed that these sequences might function to cyclize the RNA during replication (5). The cyclization sequence at the 5Ј end is located within the region encoding the N-terminal region of the capsid protein. Its 3Ј counterpart is located in the 3Ј nontranslated region. A core region of 8 nucleotides within the cyclization domain is conserved among all mosquito-borne flaviviruses (5). Khromykh et al. (7,8) examined the importance of the cyclization sequences by analyzing the ability of truncated and mutated Kunjin virus (KUNV) RNA molecules to replicate in transfected cells. They found that deletion mutants lacking the sequence encoding the first 20 amino acids of KUNV capsid protein are unable to replicate (8). They further showed that a mutant RNA that had five changes within the 8-nucleotide core conserved sequence was unable to replicate. Compensating changes in the 3Ј region that restored the complementarity of the cyclization sequence restored the ability of the RNA to replicate, althoug...

BackgroundThe plastid maturase MatK has been implicated as a possible model for the evolutionary “missing link” between prokaryotic and eukaryotic splicing machinery. This evolutionary implication has sparked investigations concerning the function of this unusual maturase. Intron targets of MatK activity suggest that this is an essential enzyme for plastid function. The matK gene, however, is described as a pseudogene in many photosynthetic orchid species due to presence of premature stop codons in translations, and its high rate of nucleotide and amino acid substitution.ResultsSequence analysis of the matK gene from orchids identified an out-of-frame alternative AUG initiation codon upstream from the consensus initiation codon used for translation in other angiosperms. We demonstrate translation from the alternative initiation codon generates a conserved MatK reading frame. We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods. We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history.ConclusionThese findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0491-1) contains supplementary material, which is available to authorized users.