Synthesis in vitro of rabbit hemorrhagic disease virus subgenomic RNA by internal initiation on (-)sense genomic RNA: mapping of a subgenomic promoter.
Rabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3' one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3'-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (-)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (-)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae.
Morales
Mónica
M
Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
Bárcena
Juan
J
Ramírez
Miguel A
MA
Boga
José A
JA
Parra
Francisco
F
Torres
Juan M
JM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D002874:Chromosome Mapping; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics*; D007313:Insecta; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D009841:Oligonucleotides; D011108:Polymers; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases