Format

Send to

Choose Destination
See comment in PubMed Commons below
Viruses. 2014 Apr 9;6(4):1637-53. doi: 10.3390/v6041637.

Generation of West Nile virus infectious clones containing amino acid insertions between capsid and capsid anchor.

Author information

1
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.
2
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.
3
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.
4
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.

Abstract

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.

PMID:
24721788
PMCID:
PMC4014714
DOI:
10.3390/v6041637
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Multidisciplinary Digital Publishing Institute (MDPI) Icon for PubMed Central
    Loading ...
    Support Center