The genus Flavivirus consists of many important arthropod-borne human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) (Gubler D., Kuno, G., Markoff, L., 2007). WNV is widely distributed throughout the world, and is found on all continents except Antarctica (Kramer L D, et al., 2008). Since the initial outbreak of WNV in New York in 1999, WNV has become an important emerging virus. So far, there is no effective antiviral therapy, and no vaccine has been approved for WNV infections.
The WNV genome is a single-stranded, positive-sense RNA molecule approximately 11 kb in length that consists of a 5′ untranslated region (UTR), a single open-reading frame (ORF) and a 3′ UTR. The ORF is translated into a polyprotein that is co-and post-translationally processed by viral and host proteases into three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Brinton M A, 2002; Mukhopadhyay S, et al., 2005). The development of antiviral agents or vaccines is of interest to control WNV infection in addition to mosquito control. However, work with WNV requires biosafety level 3 (BSL-3) facilities, which hinder research for some groups because of safety issues. Previous studies have shown that the complete viral genome RNA without the structural proteins, the replicon, has all the elements for viral replication in transfected cells, but cannot produce infectious viral particles (Shi P-Y, et al., 2002). The replicon system enables researchers to perform studies in biosafety level 2 (BSL-2) facilities. Further study showed that a foreign reporter gene [such as green fluorescent protein (GFP) and Renilla luciferase (Rluc)] could be inserted into the replicon to monitor viral replication (Lo M K, et al., 2003; Shi P-Y, et al., 2002). The Flavivirus replicons are powerful research tools to study the elements of viral replication such as the UTR, nonstructural proteins and viral encapsidation (Lo M K, et al., 2003). The replicon system is also an important tool for antiviral study and drug screening (Alvarez D E, et al., 2005; Holden K L, et al., 2006; Ng C, et al., 2007; Rossi S L, et al., 2005).
In this study, we developed and characterized a WNV replicon (Gluc-WNV-Rep) expressing the secreted Gaussia luciferase (Gluc). The culture medium of Gluc-WNV-Rep RNA-transfected cells was used for a direct assay of luciferase activity to monitor replicon replication. By using a known Flavivirus inhibitor, we showed that GlucWNV-Rep could also be used for antiviral screening.
BHK-21 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. For the generation of anti-NS4B anti-serum, BALB/c mice were immunized with a mixture of purified WNV GST-NS4B protein and complete Freund's adjuvant for the primary immunization, and then immunized with two doses of a mixture of purified WNV GST-NS4B protein and incomplete Freund's adjuvant at a 2-week interval.
As shown in Fig. 1, an infectious cDNA clone of WNV (designated as pACYC-FLWN), was used to construct a cDNA clone of a Gaussia luciferase WNV replicon (Gluc-WNV-Rep). In the replicon, the coding sequences from 421 to 2379 were replaced with Gluc-2A, while the mature capsid protein and 30 amino acids at the C-terminus of the E protein were retained.
Figure 1. WNV genome and replicons with the Gaussia luciferase reporter constructed in this study. Compared to the full-length WNV genome, the original replicon (Rep) contained an in-frame deletion of the structural region from nt 325 to 2379. The mature capsid protein and the C-terminal 30 amino acids from the envelope protein were retained to maintain the essential 5′ CS element and to ensure correct processing of the polyprotein.
A three-step cloning strategy was used to construct the Gluc-WNV-Rep plasmid. A standard overlap PCR was performed to create a cassette containing the BamHI-T7 promoter-5′ UTR-mature capsid protein, Gaussia luciferase gene, FMDV2A, and the C-terminal 30 amino acids of the E protein to the unique SmaI site in the NS2A protein. The fragment including BamHI to mature capsid was amplified with primer 1 and primer 2 using the WNV infectious clone as a template. The fragment containing the Gaussia luciferase gene was amplified with primer 3 and primer 4 using pGluc-Basic as a template. The fragment spanning the FMDV2A-C-terminal 30 amino acids of the E protein to the unique SmaI site in the NS2A protein (nucleotide position 4018 of the viral genome) was amplified with primer 5 and primer 6 using the pACYC Rluc2A WNV Rep as a template. The primer sequences are presented in Table 1. The three fragments were fused together with primer 1 and primer 6 and then digested by BamH I and Sma I. The fragment from BamH I to SmaI was engineered at the corresponding sites into pACYC-FLWN, resulting in plasmid Gluc-WNV-Rep. A non-replicative NS5 mutation GDD-AAA was introduced into the Gluc-WNV-Rep by site-directed mutagenesis, producing Gluc-WNV-Rep-mt. All the constructs were verified by DNA sequencing.
Table 1. Primers and sequences used in this study
The RNAs were in vitro transcribed from XbaI-linearized cDNA plasmids using mMESSAGE MEGAscript® T7 Kit (Ambion) according to the manufacturer's protocols. The RNAs were electroporated into BHK-21 cells as previously described by Zhang B, et al. (2010). After transfection, the supernatants and cell lysates were collected at different time points and used to measure Gluc activity.
BHK-21 cells transfected with the Gluc-WNV-Rep and Gluc-WNV-Rep-mt RNAs were seeded on chamber slides (Nalgene Nunc). At 24, 48, and 72 hours post-transfection (h p.t.), the cells were fixed with cold (−20℃) 5% acetic acid in methanol for 15 min at room temperature. The fixed cells were washed with PBS and incubated with anti-WNV-NS4B mouse polyclonal antibody (1:250 dilutions with PBS) for 1 h. After washing with PBS three times, the cells were incubated with goat anti-mouse IgG conjugated to Texas red at room temperature for 1 h. After washing three times with PBS, the slide was mounted with 90% glycerol and examined under a fluorescence microscope (Nikon). Cell images were taken at ×200 magnification.
The RNA (10 μg) of Gluc-WNV-Rep was electroporated into 8×106 BHK-21 cells. Electroporation was performed at 25 μF and 850 V with three pulses at 3-s intervals. The cells were seeded in 12-well plates and culture medium was collected at various time points for analysis. The cells were washed once with cold PBS, and 200 μL of lysis buffer (Promega) was added. The plates containing the lysis buffer were sealed with Parafilm and stored at −80℃. When samples had been collected at all time points, 20 μL of cell culture medium and cell lysate were transferred to 96-well plates. Based on the setting procedure, 50 μL BioLux Gaussia luciferase substrate (New England Biolabs, MA, USA) was primed into the sample wells by an injector (Varioskan Flash, Thermo Fisher, Finland), which were then assayed for luciferase signals with a Multimode Microplate Reader (Varioskan Flash).
BHK cells transfected with Gluc-WNV-Rep RNA were seeded into 12-well plates at a density of 3.2×105 cells per well. After 2 h, the cells were incubated with various concentrations of NITD008 (0 μmol/L to 27 μmol/L). Incubations were carried out in triplicate, and six control wells were included. The cell culture medium and cell lysates were collected at 48 h.p.t. The Gluc signal was measured with a Multimode Microplate Reader (Varioskan).