Fig 2.

Production of infectious WNV carrying a GFP reporter gene. (A) Schematics of the pWNV-GFP-backbone constructs. A variant of pWNV-backbone was constructed by introducing the GFP reporter gene between the 5′ UTR and the C gene using a strategy described previously by Shustov and colleagues (66). The first 60 nucleotides of capsid (C60) were duplicated and introduced at the amino terminus of GFP to ensure the proper cyclization and replication of pWNV-GFP-backbone. Three versions of pWNV-GFP-backbone were constructed. These three versions share the same upstream C60 at the amino terminus of GFP but are different in the downstream genomic capsid. In version 1 (V1), both upstream and downstream C60 sequences are identical. In version 2 (V2), synonymous mutations were introduced into the four amino acids comprising the cyclization sequence (CS) of the downstream capsid. In version 3 (V3), the first 60 nucleotides of the downstream capsid (including the CS) were scrambled using synonymous mutations. (B) Kinetics of WNV production using V3 of the WNV-GFP-backbone system. WNV-GFP viruses were produced as described in the legend of Fig. 1. Virus-containing supernatants were harvested at the indicated times, and titers were determined by using Raji-DC-SIGNR cells. Infection was scored as a function of GFP expression at 16 h postinfection. Data are representative of two independent experiments. (C) Comparison of the kinetics of WNV production using the three versions of the WNV-GFP-backbone system. The infectious titer of virus in supernatants harvested at the indicated hours posttransfection was calculated by using the linear portion of the virus dose-infectivity curve. Data are representative of two separate experiments. (D) Growth kinetics of WNV production using WNV-GFP-backbone V3. A virus stock produced in HEK-293T cells was used to inoculate HEK-293T, BHK-21, and C6/36 cells at a multiplicity of infection (MOI) of 0.5. Viruses were collected from infected cells at the indicated time points, and titers were determined on Raji-DC-SIGNR cells. Similar results were obtained from independent experiments performed at MOIs of 3 and 0.05.