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Overview of the Vaccinia Virus Expression System
Publication Name:
Current Protocols in Protein Science
Unit Number:
Unit 5.11
DOI:
10.1002/0471140864.ps0511s13
Online Posting Date:
May, 2001
Abstract
The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+ kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and “appropriate” transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.
Figures
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Figure 5.11.1 Replication cycle of vaccinia virus. After entry of vaccinia virus into the cells, early genes are expressed, leading to secretion of several proteins (including a growth factor), uncoating of the virus core, and the synthesis of DNA polymerase (and other replication proteins), RNA polymerase subunits, and transcriptional transactivators of the intermediate class of genes. After DNA replication, intermediate mRNAs are made, some of which encode late transcriptional transactivators. This in turn leads to expression of late genes, which encode structural proteins, enzymes, and early transcription factors that are packaged in the assembling virus particles. Some of the mature virions are wrapped in Golgi-derived membranes and are released from the cell. The bold arrows indicate products that exit the cell. Reprinted with permission from Raven Press (Moss, -
Figure 5.11.2 Flow chart showing protocols for gene expression using the recombinant vaccinia virus system.
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Literature Cited
| Literature Cited | |
| Bennink, J.R. and Yewdell, J.W. 1990. Recombinant vaccinia viruses as vectors for studying T lymphocyte specificity and function. Curr. Topics Microbiol. Immunol. 163:153-184. | |
| Baxby, D. 1989. Smallpox vaccination for investigators. Lancet 2:919. | |
| Earl, P.L., Hügin, A.W., and Moss, B. 1990. Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus. J. Virol. 64:2448-2451. | |
| Mackett, M., Smith, G.L., and Moss, B. 1982. Vaccinia virus: A selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. U.S.A. 79:7415-7419. | |
| Moss, B. 1996a. Poxviridae: The viruses and their replication. In Virology (B.N. Fields D.M. Knipe and P.M. Howley eds.) pp. 2637-2671. Raven Press, New York. | |
| Panicali, D. and Paoletti, E. 1982. Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Natl. Acad. Sci. U.S.A. 79:4927-4931. | |
| Richmond, J.Y. and McKinney, R.W. 1993. Biosafety in Microbiological and Biomedical Laboratories 3rd ed. U.S.Government Printing Office, Washington, D.C. | |
| Tartaglia, J., Perkus, M.E., Taylor, J., Norton, E.K., Audonnet, J.C., Cox, W.I., Davis, S.W., Vanderhoeven, J., Meignier, B., Riviere, M., Languet, B., and Paoletti, E. 1992. NYVAC–A highly attenuated strain of vaccinia virus. Virology 188:217-232. | |
| Wenzel, R.P. and Nettelman, M.D. 1989. Smallpox vaccination for investigators using vaccinia recombinants. Lancet 2:630-631. | |
| Key References | |
| Johnson, G.P., Goebel, S.J., and Paoletti, E. 1993. An update on the vaccinia virus genome. Virology 196:381-401. | |
| Moss, B. 1996b. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc. Natl. Acad. Sci. U.S.A. 93:11341-11348. | |
