Manual Vaccinia Virus and Poxvirology: Methods and Protocols (Methods in Molecular Biology Vol 269)

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  3. Vaccinia Virus and Poxvirology: Methods and Protocols by Humana Press Inc. (Paperback, 2010)
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Poxviruses utilize overlapping promoters as a means to modulate their activities. Poxviruses utilize host transcription factors to regulate their own gene expression.

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Virus genes 36, Bidirectional promoters in the vaccinia virus genome. Virology, , Downregulation of vaccinia virus intermediate and late promoters by host transcription factor YY1. The second project involved determination of the relationship between RNA polymerases of cellular origin and those of poxviruses. Knutson, graduate trainee Jaewook Oh, graduate trainee J. Jang, postdoctoral trainee D. Akin, collaborator, Dept. Lim, graduate trainee R. Bashir, collaborator, Dept. Ladisch, collaborator, Dept. Impacts Progress was made toward development of a device designed to detect virus particles in aerosols.

This project was a collaboration with engineering departments at Purdue. Poxvirus RNA polymerase were found to have higher than previously know similarity to comparable enzymes from cellular sources.

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This has implications for the evolution of poxvirus genomes. These viruses almost certainly acquired many of their genes from ancestral hosts. Publications Jang, J. Capture of airborne nanoparticles in swirling flows using non-uniform electrostatic fields for bio-sensor applications. Sensors and Actuators B: Chemical Virus Genes This protein is cellular transcription factor TATA binding protein. We now can begin to determine how viral proteins contribute to their gene regulation. Publications Knutson, B. The age of reverse biochemistry.

Protein Sci. This year we reported the discovery of the protein that is the central factor for activating two of the virus' classes of genes. This protein is a cellular transcription factor normally functioning to activate cellular genes. We have shown that the factor leaves the nucleus of the cell and is relocated to the cytoplasm where virus replication occurs. Based on these findings, we have formulated a model for poxvirus gene regulation in which the cellular factor recruits viral transcription factors to activate one class of genes while repressing the other.

Impacts This work demonstrates how poxviruses activate the expression of two classes of their genes and provides a framework for how the virus uses its own factors in conjunction with cellular factors. Vaccinia virus intermediate and late promoters are targeted by the TATA binding protein. We have found that a large number of proteins exit the nucleus after infection and many of which can be localized to viral replication complexes in the cytoplasm of the cell. These results indicate that cytoplasmic poxvirus have ample opportunity to utilize host nuclear proteins.

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  • Vaccinia Virus and Poxvirology.

Impacts Our findings provide important clues as to animal cell proteins required for virus replication. Publications Oh, J. This antibiotic was shown to specifically block post-replicative messenger RNA synthesis by the virus. Because of this effect, essentially no virus replication can proceed. We have identified an exceptionally strong vaccinia virus promoter. The activity of this promoter gives higher gene expression than any other promoter used in poxvirus studies.

Impacts The inhibition of vaccinia replication by distamycin A indicates that other DNA minor groove ligands may hold promise as antiviral drugs.

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The powerful vaccinia virus promoter will be useful for studies on poxviruses in general, but will also be useful as a tool in expression vectors. Vaccinia virus transcription. Broyles, S. An in vitro sysytem for studying vaccinia virus early genes. In Methods in Molecular Biology, vol. Totowa, N. In Press. Antiviral activity of distamycin A against vaccinia virus is the result of inhibition of postreplicative mRNA synthesis. We determined that the antibiotic specifically blocked the transcription of late messenger RNA. A manuscript describing theses results is in press Impacts We have identified a new type of drug that is effective in inhibiting poxviruses.

This information may be useful in designing drugs to treat smallpox and mokeypox virus infections. We have characterized the sequence requirements for poxvirus transcriptional promoters. We determined the intermediate class is much more important than previously appreciated. We have determined that the antibiotic Distamycin A had very significant antiviral activity against vaccinia virus.

Impacts These studies improve our understanding of how poxviruses express their genes. The antiviral activity of Distamycin A indicates that DNA minor groove ligands show promise as antiviral drugs and may be a useful approach toward developing drugs against other poxviruses such as smallpox.

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We are focusing on two host proteins that we believe to be responsible for initiation of expression of two classes of virus genes. Impacts Our goal is to identify potential targets for antiviral drug design. The human smallpox virus has recently emerged as threat of possible use as a biological warfare weapon. Our laboratory is interested in mechanisms of regulation of virus gene expression. We are focusing on expression of the late class of virus genes. We recently identified two cellular proteins that the virus uses to express its gene products.

These proteins are cellular transcription factors that are normally found in the cell nucleus but are redirected by virus infection to the cytoplasm where the virus is actively replicating. This change in protein trafficking requires active DNA synthesis on the part of the virus. This observation explains two facets of the biology of the virus not previously understood.

First, the requirement for DNA replication for pulling the proteins out of the nucleus explains why DNA replication is required for virus late gene expression. This requirement is universal for all DNA viruses, but in no other case is the requirement understood.

Vaccinia Virus and Poxvirology: Methods and Protocols by Humana Press Inc. (Paperback, 2010)

Our observations also might explain one mechanism contributing to cell death induced by virus infection. At least one of the proteins described above that exits the nucleus is required by all three RNA polymerases, explaining how they could be simultaneously inhibited. Impacts Our results further suggest that the snatching of the cellular proteins may explain how the virus blocks cellular gene expression which contributes to cell death after virus infection.

Impacts The focus of our research is poxviruses and how they regulate the expression of their genes. We have uncovered two cellular proteins that the virus snatches from the cell nucleus for expression of viral genes. Our results suggest that the snatching of the cellular proteins may explain how the virus blocks cellular gene expression which contributes to cell death after virus infection. Journal of Virology The Journal of Biological Chemistry.

Vaccinia Virus

Journal of Biological Chemistry , The late class of poxvirus genes is activated by an unknown mechanism, and the proteins that participate in targeting late genes for activation have not been identified. Our laboratory has identified a cellular protein that has high affinity for regulatory sites in the DNA of late viral genes. We have now purified this protein and shown it to be a novel complex of a previously identified cellular gene activator protein and another protein implicated in RNA processing.

We showed that this protein is localized to the nucleus in normal uninfected cells, but migrates to the cytoplasm where virus replication occurs during the infectious period when late genes are activated. We currently are working on expressing recombinant protein to confirm that it has the predicted activities in vitro.

As a test of this notion, we examined the sensitivity of the protein's DNA binding properties to the antibiotic distamycin A which is known to interact specifically with the minor groove of DNA. Distamycin was described as having antiviral properties over 30 years ago, but the basis for the inhibition of the virus was not known. The protein's ability was indeed highly sensitive to the antibiotic in vitro.

Because the activation of the early genes is the first step in the viral life-cycle, this indicated that the early transcription factor is the primary target for the antiviral drug. Another project which has shown promise lately is the definition of the proteins required for expression of viral late genes. We recently identified a protein that associates specifically with DNA sequences that direct the synthesis of late messenger RNA. To our surprise, the protein turned out to be nonviral in origin since it is present in uninfected cells. We are currently characterizing the protein in terms of its role in transcription of late viral genes.

Since distamycin is a minor groove ligand, this suggests that the transcription factor contacts the minor groove of the DNA helix, a property that is unusual for transcription factors. The drug was further shown to block cell to cell spread of the viral infectious cycle. The DNA binding domain of the vaccinia virus early transcription factor small subunit is an extended helicase-like motif. Acids Res. We are studying a viral gene activator protein. We have analyzed this protein from mutant viruses to determine the nature of the defect.

It was determined that the mutant proteins were impaired in their interaction with genes explaining why they do not activate gene expression. The mutant viruses were also shown to package less than normal levels of the gene activator protein suggesting that gene association is the basis for packaging the protein into virus particles.

Pox virus virology - The Poxviridae Virus Family - microbiology - part-4

The methods and protocols have been designed with the bench scientist in mind, being presented in a fashion that makes them useful for both starting and veteran poxvirus researchers. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Vaccinia Virus: Methods and Protocols serves as a valuable resource for scientists looking to bring new methods and procedures into their lab in order to make exciting discoveries that will continue to deepen our understanding of this fascinating virus family. JavaScript is currently disabled, this site works much better if you enable JavaScript in your browser.

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