Vaccinia Virus : the Unexpected Vector

In the age of Dolly and other spectacular feats of genetic manipulation, the advent of recombinant viruses as tools for microbiological research comes as no surprise. Although recombinant DNA technology has enjoyed over two decades of success, initially promising avenues such as gene therapy have been fraught with seemingly insurmountable technical difficulties. Until recently, gene correction has focused on site-directed recombination that interrupts the defective gene, or replaces it with an effective one, but this concept has yet to be realized. Current strategies are more directed toward generating gene insertion that does not disrupt any essential coding sequences. Viruses have recently become the vectors of choice for insertion of genetic material into target cells. Naturally, a lot of controversy surrounds the use of live infectious agents as vectors, especially when the vector is a member of Poxviridae and a cousin of Variola, the virus behind smallpox. So why is everyone so excited about Vaccinia virus...the unexpected vector?

A little history, perhaps . . . ?

As a member of the poxvirus family, the largest and most complex viruses known, vaccinia has relatives that have been around for centuries. Vaccinia and Viriola virus are the two best known members of the Poxviridae. Viriola major is the main culprit Figure 1 -- a very dead Ramses V (Courtesy of Dr. Alan Cann)for the devastating pathology we associate with smallpox. Smallpox first raised an ugly head in China and the Far East approximately 2000 years ago, claiming the lives of the rich and the poor without discrimination. No continent was free of smallpox as long as man, its only natural reservoir, continued to traverse the globe. The Pharaoh Ramses V (left -- A very dead Ramses; photo courtesy of Alan Cann) succumbed to smallpox in 1157 B.C. Nearly a millineum later, an estimated 3.5 million Aztecs died of small pox over two years, after Hernando Cortez introduced the scourge to America in 1520. 18th century Europe was not spared either, and incidence reached plague proportions, as well as claiming the lives of five European monarchs (Cann). In 1798, Edward Jenner demonstrated the protective effects of vaccinia virus against smallpox, isolating it initially from cows infected with cowpox (Jenner). Remarkably, thanks to Jenner for the dicovery of a preventative vaccine, and the World Health Organization (WHO) for worldwide innoculation, the last naturally occuring case of smallpox was in Somalia in 1977 (Fenner).

WHO recommended the discontinuation of smallpox vaccination in 1980 for all individuals except those investigators at special risk for poxvirus infection. In recent years, subsequent work has generated recombinant poxviruses, such as vaccinia, as tools for molecular biology, cell biology, and immunology. More controversially, the live recombinant viruses are being developed for possible gene therapy, vaccines for unrelated diseases, and for cancer immunotherapy. Vaccinia appears to be among the most promising of these.

Briefly. . .the Molecular Biology of Vaccinia

The most unusual, and perhaps technologically the most useful, feature of poxviruses is their ability to replicate in the infected cell's cytoplasm, and not nucleus. Infectious virions have a lipoprotein envelope surrounding a complex core of linear duplex DNA (approximately 200,000 bp long) connected at each end by hairpin loops. Virus encoded enzymes, a multi-subunit DNA-dependent RNA polymerase, a transcription factor, capping and methylating enzymes, and a poly(A) polymerase are all contained within the core. Vaccinia is therefore well equipped to synthesize translatable mRNA.

Figure 1. The infectious cycle of vaccinia virus (from Bernhard Moss, 1991)

After attachment and fusion with the cell membrane, approximately 100 early virus genes are transcribed by the viral RNA polymerase (fig. 1). The replicated DNA molecules serve as templates for expression of later genes. Each temporal class of genes has characteristic promoter sequences recognized by specific viral proteins. Once the late structural proteins are synthesized, the virion is assembled, and some are packaged and released with an additional Golgi-derived membrane. Vaccinia is known to undergo homologous DNA recombination naturally during replication (Nakano et al). Its genes can subsequently be mapped using marker rescue (Weir et al).

So. . .why Vaccinia? Poxviruses are generally considered as the heralders of disease. However, as tools for molecular research they provide a system for combining biochemical and genetic approaches to transcription and translation. First, almost all the necessary enzymes and factors are encoded within the poxvirus genome. Second, all genetic activity occurs within the cytoplasm, providing physical separation from corresponding host events in the nucleus. Third, early transcription components exist within the packaged core. Finally, isolating viral mutants is a relatively easy task, especially since the genome is entirely sequenced (Moss).

The Making of "the Unexpected Vector"

As stated above, vaccinia undergoes homologous recombination during replication in infected cells. When used as an expression vector, this innate ability to recombine is used to introduce foreign DNA coupled to a vaccinia promoter, such as tk, into the viral genome (fig. 2). Numerous variants already exist, including those with indicators such as the lac-Z gene for blue-white selection (Cann). The steps below outline the construction of the vaccinia expression vector (fig. 2, by permission of Alan Cann) (Moss).

(1) Your favorite gene (YFG) is flanked with vaccinia DNA sequences, especially the vaccinia promoters and multicloning sites for cleavage and ligation. The following are often included:

(2) The product (usually a plasmid with an ori and a marker gene) is then inserted into a cell infected with the whole virus. The whole virus must be used because it contains the necessary enzymes and factors within its core.

(3) Recombination during replication leads to insertion of YFG (i.e. the foreign DNA) into the viral progeny. The usual target of insertion is a nonessential region, so that virus retains its ability to replicate independently and the system can be maintained. The estimated incidence of successful insertion is approximately 0.1% (hey, I didn't say this was easy...). A major advantage of the vaccinia vector is that atleast 25,000 bp of DNA (a lot more than most vectors can handle) can be added to the vaccinia genome without requiring any deletions.

(4) Controlling when and how much of YFG is expressed is easy because the poxvirus promoter sequences control the rate and time of expression, and you can regulate which promoters are in the system. The highest yeilds of protein are generally generated with the late promoters.

(5) Virus plaques can finally be screened by DNA hybridization or for expression of your favorite protein.

With the rapid discovery of new genes, especially from the Human Genome Project, comes the daunting task of understanding how the products of these genes are synthesized, regulated, and used within cells. Vaccinia virus, as a vector for expression systems, is a powerful addition to the range of molecular methods available for such purposes. The use of Vaccinia allows temporal, as well as quantitative regulation of protein expression. More importantly, Vaccinia is large enough to accomodate several gene inserts while preserving the entire length of its DNA. Finally, as an infectious agent, it can target specific cells for insertion, and may thus be employed in gene and cancer therapy. Led by Vaccinia, the Poxviridae may no longer be considered the scourge of the world, but rather powerful tools for advancing research and therapeutic avenues.

If you would like to learn more about the current applications of Vaccinia virus, visit these sites:

References

Cann, A. <nna@le>. "Poxviruses" 12 Jan. 1998. <http://www-micro.msb.le.ac.uk/335/Poxviruses.html>

Dales, S. "Reciprocity in the interactions between poxviruses and their host cells." Annual Review of Microbiology 44 (1990):173-92.

Fenner, F., Henderson, D. A., Arita, I., Jezek, Z., & Ladnyi, I. D. Smallpox and its Eradication (World Health Organization, Geneva, 1988).

Moss, B. "Vaccinia Virus: A Tool for Research and Vaccine Development." Science 252 (1991): 1662-67.

Jenner, E., in Classics of Medicine and Surgery. C. N. B. Camac. New York: Dover, (1959). 213-40.

Nakano, E., Panicali, D., & Paoletti, E. "Molecular genetics of vaccinia virus: demonstration of marker rescue." The Proceedings of the National Academy of Sciences of the United States of America 79 (1982): 1593-96.

Unger, T. F. <tfunger@aol.com>. "Show Me The Money: Prokaryotic Expression Vectors And Purification Systems" 1 Sep. 1997. <http://www.the-scientist.library.upenn.edu/yr1997/sept/profile2_970901.html>

Weir, J. P., Bajszar, G., & Moss, B. "Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA" The Proceeding of the National Academy of Sciences of the United States of America 79 (1982): 1210-14.

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