title: Gene N proximal and distal RNA motifs regulate coronavirus nucleocapsid mRNA transcription.
abstract: Coronavirus subgenomic mRNA (sgmRNA) transcription requires a discontinuous RNA synthesis mechanism driven by the transcription-regulating sequences (TRSs), located at the 3' end of the genomic leader (TRS-L) and also preceding each gene (TRS-B). In transmissible gastroenteritis virus (TGEV), the free energy of TRS-L and cTRS-B (complement of TRS-B) duplex formation is one of the factors regulating the transcription of sgmRNAs. In addition, N gene sgmRNA transcription is controlled by a transcription-regulating motif, including a long-distance RNA-RNA interaction between complementary proximal and distal elements. The extension of complementarity between these two sequences increased N gene transcription. An active domain, a novel essential component of the transcription-regulating motif, has been identified. The active domain primary sequence was necessary for its activity. Relocation of the active domain upstream of the N gene TRS core sequence in the absence of the proximal and distal elements also enhanced sgmRNA N transcription. According to the proposed working model for N gene transcriptional activation, the long-distance RNA-RNA interaction relocates the distant active domain in close proximity with the N gene TRS, which probably increases the frequency of template switching during the synthesis of negative RNA. The transcription-regulating motif has been optimized to a minimal sequence showing a 4-fold activity increase in relation to the native RNA motif. Full-length TGEV infectious viruses were generated with the optimized transcription-regulating motif, which enhanced by 5-fold the transcription of the 3a gene and can be used in expression vectors based in coronavirus genomes.
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, Darwin 3, Campus de la Universidad Autonoma de Madrid, 28049 Madrid, Spain.
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Mateos-Gómez
authors.forename: Sonia
authors.initials: S
authors.lastname: Zuñiga
authors.forename: Lorena
authors.initials: L
authors.lastname: Palacio
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
authors.forename: Isabel
authors.initials: I
authors.lastname: Sola
mesh_terms: D020029:Base Pairing; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins / Q000096:biosynthesis*; D018075:RNA, Complementary / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics*; D014158:Transcription, Genetic; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000502:physiology*
chemical_list: D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D018075:RNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral