सरल और मजबूत Vivo में और इन विट्रो में दृष्टिकोण वायरस विधानसभा अध्ययन के लिए
Summary
एक सरल, कुशल और मजबूत करने के लिए संयंत्र कोशिकाओं के माध्यम से कई वायरल घटकों का वितरण सिंक्रनाइज़<em> Agrobacterium</em> मध्यस्थता क्षणिक अभिव्यक्ति वर्णित है. यह दृष्टिकोण प्रतिकृति अध्ययन के लिए उत्तरदायी है, encapsidation द्वारा पीछा<em> इन विट्रो में</em> जीनोम में गैर वायरल घटकों के पुनः समवेत ऑप्टिकल वायरल जैव चिकित्सा अनुप्रयोगों के लिए उपयुक्त भूत समाप्त.
Abstract
In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.
Protocol
Discussion
यहाँ प्रस्तुत agroinfiltration दृष्टिकोण व्यापक रूप से संयंत्र वायरस की एक विस्तृत श्रृंखला के लिए लागू किया जा सकता है. इस दृष्टिकोण की एक बानगी की सुविधा एक ही सेल एक प्रमुख दोष आमतौर पर नियमित रूप से इस्तेमाल ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
लेखकों उनके बहुमूल्य सुझावों के लिए agroinfiltration के विकास में और इन विट्रो विधानसभा assays में प्रयोगशाला के कई सदस्यों को धन्यवाद देना चाहूंगा. इस काम कैलिफोर्निया विश्वविद्यालय से एक अनुदान द्वारा वित्त पोषित किया गया था. इस अध्ययन भागों में राष्ट्रीय विज्ञान फाउंडेशन (CBET 1,144,237) से एक अनुदान द्वारा समर्थित किया गया.
Materials
| Name of the reagent | Company | Catalog number |
| MES Sodium salts | Sigma-aldrich | M2993 |
| Indocyanine green | Sigma-aldrich | I2633 |
| Beckman ultracentrifuge | Beckman Coulter | Model: L8-70M |
| Centricon-100 column | Millipore | YM-100 |
| Spectrophotometer | Cary 50, Varian Inc. | Part number 10068900 |
| Spectrofluorometer | Fluorolog 3, Jobin-Yvon. | Part number FL3-21 |
Riferimenti
- Annamalai, P., Rao, A. L. Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging. Virology. 338, 96-111 (2005).
- Annamalai, P., Rofail, F., Demason, D. A., Rao, A. L. Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration. J. Virol. 82, 1484-1495 (2008).
- Annamalai, P., Rao, A. L., Foster, N., Johansen, H. . Plant Virology Protocols. 451, 251-264 (2008).
- Lane, L. C. The bromoviruses. Adv. Virus Res. 19, 151-220 (1974).
- Choi, Y. G., Rao, A. L. Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals. Virology. 275, 207-217 (2000).
- Sambrrok, J., Russel, D. W. . Molecular Cloning. A Laboratory Manual. , (2001).
- Dreher, T. W., Rao, A. L., Hall, T. C. Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation. J. Mol. Biol. 206, 425-438 (1989).
- Jung, B., Rao, A. L., Anvari, B. Optical nano-constructs composed of genome-depleted brome mosaic virus doped with a near infrared chromophore for potential biomedical applications. A.C.S. Nano. 5, 1243-1252 (2011).
- Voinnet, O., Lederer, C., Baulcombe, D. C. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell. 103, 157-167 (2000).
- Wroblewski, T., Tomczak, A., Michelmore, R. Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol. J. 3, 259-273 (2005).
