Identification of specific nucleotide sequences within the conserved 3'-SL in the dengue type 2 virus genome required for replication.
The flavivirus genome is a positive-stranded approximately 11-kb RNA including 5' and 3' noncoding regions (NCR) of approximately 100 and 400 to 600 nucleotides (nt), respectively. The 3' NCR contains adjacent, thermodynamically stable, conserved short and long stem-and-loop structures (the 3'-SL), formed by the 3'-terminal approximately 100 nt. The nucleotide sequences within the 3'-SL are not well conserved among species. We examined the requirement for the 3'-SL in the context of dengue virus type 2 (DEN2) replication by mutagenesis of an infectious cDNA copy of a DEN2 genome. Genomic full-length RNA was transcribed in vitro and used to transfect monkey kidney cells. A substitution mutation, in which the 3'-terminal 93 nt constituting the wild-type (wt) DEN2 3'-SL sequence were replaced by the 96-nt sequence of the West Nile virus (WN) 3'-SL, was sublethal for virus replication. An analysis of the growth phenotypes of additional mutant viruses derived from RNAs containing DEN2-WN chimeric 3'-SL structures suggested that the wt DEN2 nucleotide sequence forming the bottom half of the long stem and loop in the 3'-SL was required for viability. One 7-bp substitution mutation in this domain resulted in a mutant virus that grew well in monkey kidney cells but was severely restricted in cultured mosquito cells. In contrast, transpositions of and/or substitutions in the wt DEN2 nucleotide sequence in the top half of the long stem and in the short stem and loop were relatively well tolerated, provided the stem-loop secondary structure was conserved.
Laboratory of Vector-Borne Virus Diseases, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA.
L
L
Zeng
B
B
Falgout
L
L
Markoff
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D003716:Dengue Virus / Q000235:genetics* / Q000502:physiology; D001330:Electronic Data Processing; D016679:Genome, Viral; D007700:Kinetics; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012367:RNA, Viral / Q000737:chemistry; D013329:Structure-Activity Relationship; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000032:analysis; D014779:Virus Replication; D014902:West Nile virus / Q000235:genetics
D012367:RNA, Viral; D014764:Viral Proteins