A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication
Abstract
:1. Introduction
2. Results
2.1. System for Production of RVF-VLPs from ClonedcDNAs
Plasmid name | Description | Ref |
---|---|---|
pTrRVFV-SDNSs::GFP | S segment-based minigenomeplasmid. The NSs gene has been replaced with GFP. Primary transcription is mediated by T7 RNAP. Vector backbone derived from pSP64 (Promega). | [16] |
pRdRp | RdRp ORF cloned into pVAX1 (Invitrogen). Contains T7 RNAP and CMV promoters. | This study |
pRdRp-Amp (pSRG309) | RdRp ORF cloned into pIRES (Clontech). Contains T7 RNAP and CMV promoters. | This study |
pN | N ORF cloned into pVAX1 (Invitrogen). Contains T7 RNAP and CMV promoters. | This study |
pSTrRVFV-SDNSs::hRLuc | S segment-based minigenomeplasmid. The NSs gene has been replaced with hRLuc. Primary transcription is mediated by T7 RNAP. Vector backbone is pSMART HC Kan (Lucigen). | This study |
pSTrRVFV-SDNDNSs::hRLuc | Derived from pSTrRVFV-SDNSs::hRLuc, the SmaI fragment within the N gene has been removed. | This study |
pGn/Gc | Gn/Gcpolyprotein ORF cloned into pcDNA1.1 | [22] |
2.2. Production of Infectious RVF-VLPs.
Sample | Average Log Raw Luciferase Units (RLU) |
---|---|
EV/EV | 3.76 |
RdRp/EV | 6.94 |
RdRp/Gn/Gc | 8.37 |
VLP Harvest (hpt) | Sample | BSR-T7/5 (Log RLU/mL) | BSR-T7/5 RdRp/N-expressing (Log RLU/mL) | Vero (Log RLU/mL) | Vero RdRp-expressing (Log RLU/mL) |
---|---|---|---|---|---|
24 | EV/EV | 3.21 | 3.71 | 3.22 | 3.24 |
RdRp/EV | 3.25 | 3.55 | 3.22 | 3.23 | |
RdRp/Gn/Gc | 5.38 | 6.36 | 4.50 | 4.93 | |
48 | RdRp/EV | 3.80 | 4.42 | 3.69 | 3.70 |
RdRp/Gn/Gc | 7.08 | 8.31 | 6.15 | 6.41 | |
72 | RdRp/EV | 3.28 | 4.01 | 3.23 | 3.26 |
RdRp/Gn/Gc | 6.04 | 7.54 | 5.10 | 5.37 |
2.3. RVF-VLPs are Efficiently Produced
2.4. RVF-VLPs are Antigenically Indistinguishable from Authentic RVFV
2.5. RVF-VLPs are Efficiently Harvested by High-Speed Centrifugation
2.6. RVF-VLPsBehave Similar to Authentic RVFV with Respect to Small Molecule Inhibitors
3. Discussion
Inhibitor | Concentration | % Activity |
---|---|---|
Mock (water) | - | 100 |
Ammonium chloride | 5 mM | 9.7 |
10 mM | 1.3 | |
20 mM | 0.5 | |
Guanidine | 25 mg/mL | 45.8 |
50 mg/mL | 19.2 | |
100 mg/mL | 22.4 | |
Ribavirin | 25 mg/mL | 12.8 |
50 mg/mL | 6.2 | |
100 mg/mL | 4.3 | |
Mock (ethanol) | - | 100 |
Actinomycin D | 0.5 mg/mL | 13.2 |
1.0 mg/mL | 6.4 | |
2.0 mg/mL | 5.2 | |
Monensin | 0.1 mM | 58.2 |
1.0 mM | 2.1 | |
10.0 mM | 0.2 | |
Mycophenolic acid | 1.0 mM | 48.6 |
10.0 mM | 8.9 | |
100.0 mM | 5.8 |
4. Experimental Section
4.1. Plasmids
4.2. Cells and Virus
4.3. Antibodies
4.4. Virus-Like Particle Production
4.5. RVFV Inhibitor Screen
4.6. Immunofluorescence Microscopy
5. Conclusions
Acknowledgments
References
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Piper, M.E.; Gerrard, S.R. A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication. Viruses 2010, 2, 731-747. https://doi.org/10.3390/v2030731
Piper ME, Gerrard SR. A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication. Viruses. 2010; 2(3):731-747. https://doi.org/10.3390/v2030731
Chicago/Turabian StylePiper, Mary E., and Sonja R. Gerrard. 2010. "A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication" Viruses 2, no. 3: 731-747. https://doi.org/10.3390/v2030731