Influenza A virus encoding secreted Gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins.

Nadine Eckert,Florian Wrensch,Sabine Gärtner,Michael Winkler,Ulrike Goedecke,Navaneethan Palanisamy,Nils Jäger,Stefan Pöhlmann

doi:10.1371/journal.pone.0097695

Nadine Eckert, Florian Wrensch + Show 6 more

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https://doi.org/10.1371/journal.pone.0097695

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Journal: PloS one	Publication Date: May 19, 2014
Citations: 105	License type: CC BY 4.0

Affiliation: German Primate Center

Abstract

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.

Highlights

Influenza A viruses are major human pathogens causing annual epidemics and intermittent pandemics with potentially devastating consequences [1]
An elegant approach to the design of replication competent reporter viruses was used by Manicassamy and colleagues [14] who inserted the green fluorescent protein (GFP) reporter into segment 8 flanked by complete NS1 and NEP/NS2 genes
The ccdB gene acts as a toxin that kills E. coli by interfering with DNA gyrase activity [22,23]

Summary

Introduction

Influenza A viruses are major human pathogens causing annual epidemics and intermittent pandemics with potentially devastating consequences [1]. The addition of reporter genes to the genome should generate a viable and stable virus able to replicate in the same spectrum of target cells as the corresponding wild type (wt) virus. Several such replication-competent influenza A viruses carrying reporter genes have been described to date [11,12,13]. The genes were arranged as a single open reading frame encoding the NS1-GFP fusion protein connected to NEP/NS2 via a 2A StopGo-sequence, which terminates translation followed by re-initiation [15] This strategy circumvented the duplication of packaging signals, but required mutation of the splice acceptor signal to prevent splicing of this segment. For quantitation in cellbased systems, enzymatic reporter assays are more sensitive

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