title: Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3' stem-loop structure is not required for replication.
abstract: The pentanucleotide (PN) sequence 5'-CACAG-3' at the top of the 3' stem-loop structure of the flavivirus genome is well conserved in the arthropod-borne viruses but is more variable in flaviviruses with no known vector. In this study, the sequence requirements of the PN motif for yellow fever virus 17D (YFV) replication were determined. In general, individual mutations at either the second, third or fourth positions were tolerated and resulted in replication-competent virus. Mutations at the fifth position were lethal. Base pairing of the nucleotide at the first position of the PN motif and a nucleotide four positions downstream of the PN (ninth position) was a major determinant for replication. Despite the fact that the majority of the PN mutants were able to replicate efficiently, they were outcompeted by parental YFV-17D virus following repeated passages in double-infected cell cultures. Surprisingly, some of the virus mutants at the first and/or the ninth position that maintained the possibility of forming a base pair were found to have a similar fitness to YFV-17D under these conditions. Overall, these experiments suggest that YFV is less dependent on sequence conservation of the PN motif for replication in animal cells than West Nile virus. However, in animal cell culture, YFV has a preference for the wt CACAG PN sequence. The molecular mechanisms behind this preference remain to be elucidated.
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands.
authors.forename: Patrícia A G C
authors.initials: PAGC
authors.lastname: Silva
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands.
authors.forename: Richard
authors.initials: R
authors.lastname: Molenkamp
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Dalebout
authors.affiliation: Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Charlier
authors.affiliation: Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium.
authors.forename: Johan H
authors.initials: JH
authors.lastname: Neyts
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands.
authors.forename: Willy J M
authors.initials: WJM
authors.lastname: Spaan
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Bredenbeek
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D006224:Cricetinae; D016679:Genome, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication; D014902:West Nile virus / Q000235:genetics; D015005:Yellow fever virus / Q000235:genetics* / Q000502:physiology*
chemical_list: D020413:3' Untranslated Regions; D012367:RNA, Viral