Generation of West Nile virus infectious clones containing amino acid insertions between capsid and capsid anchor.
West Nile virus (WNV) is a positive-sense RNA arbovirus responsible for recent outbreaks of severe neurological disease within the US and Europe. Large-scale analyses of antiviral compounds that inhibit virus replication have been limited due to the lack of an adequate WN reporter virus. Previous attempts to insert a reporter into the 3' untranslated region of WNV generated unstable viruses, suggesting that this region does not accommodate additional nucleotides. Here, we engineered two WNV infectious clones containing insertions at the Capsid (C)/Capsid Anchor (CA) junction of the viral polyprotein. Recombinant viruses containing a TAT(1-67) or Gaussia Luciferase (GLuc) gene at this location were successfully recovered. However, rapid loss of most, if not all, of the reporter sequence occurred for both viruses, indicating that the reporter viruses were not stable. While the GLuc viruses predominantly reverted back to wild-type WNV length, the TAT viruses retained up to 75 additional nucleotides of the reporter sequence. These additional nucleotides were stable over at least five passages and did not significantly alter WNV fitness. Thus, the C/CA junction of WNV can tolerate additional nucleotides, though insertions are subject to certain constraints.
Maryland Pathogen Research Institute and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Prince George's County, MD 20742, USA. rvanderg@umd.edu.
Rianna
R
Vandergaast
Maryland Pathogen Research Institute and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Prince George's County, MD 20742, USA. linjaian@umd.edu.
Lisa I
LI
Hoover
Maryland Pathogen Research Institute and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Prince George's County, MD 20742, USA. kzheng@umd.edu.
Kang
K
Zheng
Maryland Pathogen Research Institute and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Prince George's County, MD 20742, USA. brenda.fredericksen@nih.gov.
Brenda L
BL
Fredericksen
D036022:Capsid Proteins / Q000235:genetics*; D017930:Genes, Reporter; D042822:Genomic Instability; D008156:Luciferases / Q000235:genetics; D016254:Mutagenesis, Insertional; D059386:Reverse Genetics; D014902:West Nile virus / Q000235:genetics* / Q000502:physiology*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics
D036022:Capsid Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D008156:Luciferases