Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

Details

• Alternative Title:
  Virus Res
• Personal Author:
  Leardkamolkarn, Vijittra ; Sirigulpanit, Wipawan ; Chotiwan, Nunya ; Kumkate, Supeecha ; Huang, Claire Y.-H.
• Description:
  Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a full-length DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5'noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.
• Subjects:
  5′ NCR Mutation Article Dengue Virus Dengue Virus Type 2 DENV/GFP Plasmid Genes, Reporter Green Fluorescent Proteins Humans Recombination, Genetic Replicon RNA, Viral Staining And Labeling Subgenomic Replicons Virology Virus Replication

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• Source:
  Virus Res. 163(2):552-562
• Pubmed ID:
  22197424
• Pubmed Central ID:
  PMC4582755
• Document Type:
  Journal Article
• Funding:
  CC999999/Intramural CDC HHSUnited States/
• Volume:
  163
• Issue:
  2
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:ab1b96b950e57d803a07b52ebab91e99f7921fe1ff37d73956db121bac786534
• Download URL:
  https://stacks.cdc.gov/view/cdc/34606/cdc_34606_DS1.pdf
• File Type:
  [PDF - 2.93 MB ]