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

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.

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

“…However, the instability or decreased fitness of the recombinant viruses compared to the parental virus limit their application in some experimental designs, as noted by Aubry et al (2015). This instability in engineered virus expressing reporter genes was also reported in other flavivirus and DNA virus platforms (Song et al 2012, Vandergaast et al 2014, Zhang et al 2015.…”

“…Zhang et al (2015) reported the construction of a West Nile Virus (WNV) expressing GLuc and its use in neutralization assays, but the recombinant virus gradually lost stability along the passages, where GLuc activity was no longer detected in passage 3. Vandergaast et al (2014) also found similar instability in a GLuc-expressing WNV. The lower stability of these recombinant viruses in relation to YFV-GLuc probably is due to the insertion position GLuc gene (within C gene) and/or to the pathogenic strain used as backbone of WNV-GLuc, especially in Vandergaast et al (2014).…”

“…Vandergaast et al (2014) also found similar instability in a GLuc-expressing WNV. The lower stability of these recombinant viruses in relation to YFV-GLuc probably is due to the insertion position GLuc gene (within C gene) and/or to the pathogenic strain used as backbone of WNV-GLuc, especially in Vandergaast et al (2014). Conversely, in our experiments, GLuc gene was inserted into E-NS1 junction of an attenuated virus (YFV-17D).…”

Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay

“…However, none of a stable luciferase protein-based WNV reporter virus has been available for measuring neutralizing antibody, though several WNV reporter viruses were developed (Deas et al, 2005;McGee et al, 2010;Pierson et al, 2005;Vandergaast et al, 2014). In this study, we have developed a stable WNV reporter virus using Gaussia luciferase as a reporter gene, which has a higher enzymatic activity compared with Renilla luciferase (Tannous et al, 2005).…”

Development of viable TAP-tagged dengue virus for investigation of host–virus interactions in viral replication

“…passages (Vandergaast et al, 2014). Similarly, a C-terminal GFP tag of Sindbis virus's RdRp failed to infect cells, but virus was viable when the tag was switched to 3|FLAG (Cristea et al, 2010).…”