रिवर्स आनुवंशिकी का उपयोग करने के लिए दरार घाटी बुखार वायरस सांसद 12 वैक्सीन सुरक्षा और प्रभावकारिता में सुधार तनाव के एनएसएस जीन हेरफेर
Summary
रिवर्स आनुवंशिकी प्रणाली दरार घाटी बुखार वायरस सांसद 12 टीका तनाव के लिए बढ़ क्षीणन और प्रतिरक्षाजनकता साथ अतिरिक्त सांसद 12 म्यूटेंट बनाने के लिए एक उपयोगी उपकरण है. हम प्रोटोकॉल का वर्णन करने के लिए पैदा करते हैं और एनएसएस उत्परिवर्ती उपभेदों विशेषताएँ.
Abstract
Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.
Protocol
Discussion
RVFV के लिए आनुवंशिकी सिस्टम उल्टा T7 प्रमोटर 2,4,5 या 3 माउस या मानव 4 प्रमोटर नीति मैं का उपयोग करके कई समूहों द्वारा विकसित किया गया है . इस पांडुलिपि में, हम BHK/T7-9 15 कि stably व्यक्त T7 शाही सेना पोलीम?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम अनुदान 5 नंबर U54-07 उत्कृष्टता के पश्चिमी क्षेत्रीय केंद्र (WRCE) के माध्यम से AI057156, नेशनल इंस्टीट्यूट ऑफ एलर्जी और संक्रामक रोगों से 1 AI08764301 – A1 R01, और विश्वविद्यालय में एक टीके के विकास के लिए Sealy केंद्र से आंतरिक धन के द्वारा वित्त पोषित किया गया था टेक्सास चिकित्सा शाखा है.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Minimum Essential Medium (MEM)-alpha | Invitrogen | 32561037 | |
| Dulbecco’s modified minimum essential medium | Invitrogen | 11965092 | |
| Modified Eagle Medium (MEM 2x) | Invitrogen | 11935046 | |
| Penicillin-Streptomycin | Invitrogen | 15140122 | |
| Hygromycin B | Cellgro | 30-240-CR | |
| Tryptose phosphate broth | MP biomedicals | 1682149 | |
| Noble agar | VWR | 101170-362 | |
| TransIT-LT1 | Mirus | MIR2300 | |
| Opti-MEM | Invitrogen | 31985070 | |
| Aerosol tight lid | Eppendorf | C-2223-25 | |
| 0.33% neutral red solution | Sigma Aldrich | N2889-100ML | |
| C57/WT MEF cells | InvivoGen | mef-c57wt | |
| Blasticidin S | InvivoGen | Ant-bl-1 | |
| Zeocin | InvivoGen | ant-zn-1 | |
| QUANTI-Blue | InvivoGen | rep-qb1 | |
| BHK/T7-9 cells15 | Gifu university, Japan | ||
| Vero E6 cells | ATCC | CRL-1586 |
References
- Bird, B. H., Ksiazek, T. G., Nichol, S. T., Maclachlan, N. J. Rift Valley fever virus. J. Am. Vet. Med. Assoc. 234, 883-893 (2009).
- Ikegami, T., Won, S., Peters, C. J., Makino, S. Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene. J. Virol. 80, 2933-2940 (2006).
- Billecocq, A. RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses. Virology. 378, 377-384 (2008).
- Habjan, M., Penski, N., Spiegel, M., Weber, F. T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus. J. Gen. Virol. 89, 2157-2166 (2008).
- Gerrard, S. R., Bird, B. H., Albarino, C. G., Nichol, S. T. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection. Virology. 359, 459-465 (2007).
- Billecocq, A. NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription. J. Virol. 78, 9798-9806 (2004).
- May, N. L. e. TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus. Cell. 116, 541-550 (2004).
- Ikegami, T. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation. PLoS Pathog. 5, e1000287-e1000287 (2009).
- Habjan, M. NSs protein of Rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase. J. Virol. 83, 4365-4375 (2009).
- Garcia-Sastre, A., Biron, C. A. Type 1 interferons and the virus-host relationship: a lesson in detente. Science. 312, 879-882 (2006).
- Bon, A. L. e. Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity. 14, 461-470 (2001).
- Le Bon, A., Tough, D. F. Links between innate and adaptive immunity via type I interferon. Curr. Opin. Immunol. 14, 432-436 (2002).
- Tough, D. F. Type I interferon as a link between innate and adaptive immunity through dendritic cell stimulation. Leuk. Lymphoma. 45, 257-264 (2004).
- Ito, N. Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system. Microbiol. Immunol. 47, 613-617 (2003).
- Terasaki, K., Murakami, S., Lokugamage, K. G., Makino, S. Mechanism of tripartite RNA genome packaging in Rift Valley fever virus. Proc. Natl. Acad. Sci. U.S.A. 108, 804-809 (2010).
- Buchholz, U. J., Finke, S., Conzelmann, K. K. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J. Virol. 73, 251-259 (1999).
- Diaz, M. O. Homozygous deletion of the alpha- and beta 1-interferon genes in human leukemia and derived cell lines. Proc. Natl. Acad. Sci. U.S.A. 85, 5259-5263 (1988).
- Mosca, J. D., Pitha, P. M. Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis. Mol. Cell. Biol. 6, 2279-2283 (1986).
- Constantinescu, S. N. Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex. Proc. Natl. Acad. Sci. U.S.A. 92, 10487-10491 (1995).
- Kumar, K. G., Tang, W., Ravindranath, A. K., Clark, W. A., Croze, E., Fuchs, S. Y. SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor. EMBO J. 22, 5480-5490 (2003).
- Kakach, L. T., Suzich, J. A., Collett, M. S. Rift Valley fever virus M segment: phlebovirus expression strategy and protein glycosylation. Virology. 170, 505-510 (1989).
- Kakach, L. T., Wasmoen, T. L., Collett, M. S. Rift Valley fever virus M segment: use of recombinant vaccinia viruses to study Phlebovirus gene expression. J. Virol. 62, 826-833 (1988).
- Niwa, H., Yamamura, K., Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199 (1991).
- Muller, R. Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment. Am. J. Trop. Med. Hyg. 53, 405-411 (1995).
- Le May, N. A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells. PLoS Pathog. 4, e13-e13 (2008).
- Kalveram, B., Lihoradova, O., Ikegami, T. NSs Protein of Rift Valley Fever Virus Promotes Post-Translational Downregulation of the TFIIH Subunit p62. J. Virol. 85, 6234-6243 (2011).
- Taniguchi, T., Ogasawara, K., Takaoka, A., Tanaka, N. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 19, 623-655 (2001).
- Marie, I., Durbin, J. E., Levy, D. E. Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7. EMBO J. 17, 6660-6669 (1998).
- Ikegami, T., Won, S., Peters, C. J., Makino, S. Rift Valley fever virus NSs mRNA is transcribed from an incoming anti-viral-sense S RNA segment. J. Virol. 79, 12106-12111 (2005).
- Mims, C. A. Rift Valley Fever virus in mice. I. General features of the infection. Br. J. Exp. Pathol. 37, 99-109 (1956).
- Bouloy, M. Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs. J. Virol. 75, 1371-1377 (2001).
- Bird, B. H., Albarino, C. G., Nichol, S. T. Rift Valley fever virus lacking NSm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease. Virology. 362, 10-15 (2007).
