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Viruses 2014, 6(4), 1637-1653; doi:10.3390/v6041637
Article

Generation of West Nile Virus Infectious Clones Containing Amino Acid Insertions Between Capsid and Capsid Anchor

,
,
 and †,*
Maryland Pathogen Research Institute and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Prince George's County, MD 20742, USA Present address: Aids Review Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD 20817, USA.
* Author to whom correspondence should be addressed.
Received: 4 February 2014 / Revised: 19 March 2014 / Accepted: 21 March 2014 / Published: 9 April 2014
(This article belongs to the Section Viruses of Plants, Fungi and Protoza)
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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.
Keywords: West Nile virus; WNV; infectious clone; reporter; virus replication West Nile virus; WNV; infectious clone; reporter; virus replication
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Vandergaast, R.; Hoover, L.I.; Zheng, K.; Fredericksen, B.L. Generation of West Nile Virus Infectious Clones Containing Amino Acid Insertions Between Capsid and Capsid Anchor. Viruses 2014, 6, 1637-1653.

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