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- Worms in space |
Viruses, including both the viruses of eukaryotic cells and the bacteriophages of bacteria and archaea, have long served as experimental tools for prying out the deepest biological secrets of their cellular hosts. Viruses are also fascinating and informative in their own right. Viruses of both the eukaryotic and prokaryotic persuasions provide marvelous opportunities, once we understand how they work, for manipulating or modifying their hosts, as for example in gene therapy, disease detection, and other biotechnological applications. Members of the Biological Sciences Department are exploiting all of these advantages of viruses. Dr. Pipas uses the DNA tumor virus SV40, and particularly the virus-encoded tumor antigen, to ask questions about tumorigenesis and cell signaling pathways in mammalian cells. Dr. Hatfull uses the bacteriophages of Mycobacterial cells as tools to learn about pathogenesis in tuberculosis and leprosy. He has also developed phage-based approaches to detect rapidly the presence of Mycobacterium tuberculosis and to determine its drug sensitivities, as well as other methods to facilitate vaccine design. Dr. Hendrix uses phages, including bacteriophage HK97, to investigate one of the fundamental questions of biology: how is biological structure generated from its macromolecular parts? Together, Drs. Hatfull and Hendrix, as codirectors of the Pittsburgh Bacteriophage Institute, are investigating the evolution and population biology of phages using genomic techniques. Dr. Peebles, who spends most of his time thinking about RNA splicing, has been unable to control his fascination with phages and is collaborating with Dr. Hatfull on some mycobacteriophage projects. SV40 large tumor antigen, the Most Amazing Molecule in the Universe
Many viral genomes are too small to encode all of the proteins necessary for replication. This means that viruses must borrow cellular proteins in order to produce progeny virus successfully. For example, the DNA tumor virus, Simian Virus 40 (SV40), cannot replicate it's genome without usurping the DNA replication machinery of the infected cell. Viral genomes which fail to manipulate host cells successfully are simply not replicated and therefore lost from the "viral gene pool". Under this selective pressure, each viral protein has survived the ultimate test of "functional genomics"- that of natural selection. The Pipas lab makes use of one such viral protein, the SV40 Large Tumor Antigen, to trace and study key cellular regulatory systems. The diversity of cellular targets and manipulative strategies employed by the SV40 Large Tumor Antigen has led to it's unbiased and universally accepted designation as the most amazing molecule in the universe. After infecting resting cells, one of the immediate requirements of SV40 is that of overcoming normal molecular restraints upon cell division. SV40 T-antigen meets this requirement by interacting with proteins involved in controlling cell division; including the prototypic tumor suppresser proteins pRb and p53. In fact, when SV40 T-antigen is expressed in an experimental animal, tumors are formed. Changes leading to tumorigenesis can also be studied in cultured cells as shown in Figure 1. One aspect of oncogenic transformation seen in cultured cells is the loss of contact inhibition. Normal cells (Panel A) reach a certain density and then are signaled to stop growing by some as-yet unknown signal. Panel B shows cells identical to those in Panel A except that a gene for SV40 T-antigen has been introduced. These cells reach a much higher density than the normal cells seen in Panel A. When only a piece of the SV40 T-antigen protein is expressed (Panel C), cells appear to be contact-inhibited to the same extent as normal cells. This observation is fascinating because the piece of SV40 T-antigen expressed in Panel C retains regions of the protein which are known to interact with pRb. Thus, SV40 T-antigen and it's derivative protein fragments has shown that the simple binding of tumor-suppresser pRb is not enough to achieve the high cultured cell density. Using the Laser Scanning Confocal Microscope here in the Department of Biological Sciences, it was shown that full-length SV40 T-antigen allows cells to stack up in a layer after reaching confluence (Panels D-F). In this experiment, the cells shown in Panels A-C were grown past confluence and stained with a fluorescent dye. Using the confocal microscope, optical slices of the cells were made and a computer-generated 3D image of the cells was made. By rotating the image 90 degrees about the X-axis (i.e. into the plane of the picture), it can be seen that full length SV40 T-antigen permits cells to stack while the SV40 T-antigen fragment (Panel E) leaves cells in a normal-looking (compare to Panel D) monolayer. Further research has shown that cells expressing the SV40 T-antigen fragment are, in fact, different from normal cells. They continue to divide after reaching confluence but float off of the culture dish and into the culture medium rather than sticking to the culture monolayer as do cells expressing full-length SV40 T-antigen. These results are important when considering what occurs at the cellular level during the formation of a solid tumor or during metastasis. As can be seen above, mutants and fragments of SV40 T-antigen which fail to interact with pRb, p53 and other host cell proteins are useful tools for the study of pathways involving those proteins. The Pipas lab has established the SV40 Mutant Large T Antigen Database; this database represents the first of it's kind and continues to be maintained here in the Department of Biological Sciences. Luciferase reporter phages, a powerful tool for controlling the deadliest infectious disease of humankind
Diagnosis of tuberculosis (TB) is complicated by the extremely slow growth of the infectious agent, Mycobacterium tuberculosis. The clinical microbiology of TB has become yet more problematic in recent years due to the increasing prevalence of drug-resistance and multiple drug resistant strains (MDR) of M. tuberculosis. There is, therefore, a substantial need for rapid diagnostic methods that identify drug-susceptibility profiles of clinical isolates of M. tuberculosis. Luciferase reporter mycobacteriophages hold considerable promise as a novel rapid drug susceptibility test for M. tuberculosis. These phages are constructed as recombinant derivatives of well-characterized mycobacteriophages such as L5, D29, or TM4, carrying the luciferase gene from fireflies (FFlux). When these reporter phages are used to infect cultures of mycobacteria, the luciferase gene is expressed and (when a luciferin substrate is added) the infected cells emit light. This light can be readily detected using either a luminometer or photographic film. Since the background level of light emission is extremely low, the sensitivity is high, and relatively few numbers of cells can be detected in a short period of time. Production of light is dependent on the cells being alive, both because the reporter gene must be expressed and because photon production requires intracellular ATP. The assay can therefore be readily applied to testing drug susceptibility. For example, if a drug such as rifampicin is added to a sensitive strain of M. tuberculosis, then the drug will inhibit luciferase production and little or no light will be generated. However, if the strain if resistant to rifampicin then light production is will similar to the level when the drug is not added. This technology thus has the promise to deliver a rapid diagnostic test that can empirically determine the drug-susceptible profiles of clinical isolates of M. tuberculosis. To enable the use of this technology in the developing world, we have described a rather simple device, coined the "Bronx Box" that can be used to detect light production from microtitre plates. By optimizing the conditions for light production, drug-susceptibility profiles can be generated in the Bronx Box in a reasonable time-frame and without the need for complex instrumentation. This work was done in collaboration with Drs. Jacobs and Chan at Albert Einstein College of Medicine, New York. Genomic sequencing of phage L5, D29 and TM4 was carried out at the Pittsburgh Bacteriophage Institute Genome Center. Bacteriophage HK97 head assembly, a protein ballet
The Hendrix group is using bacteriophage HK97 to learn about assembly of a complex biological structure. Keywords here are: capsids, cleavage, comprehensive covalent crosslinking, cryo-EM, chainmail, crystallography, conformational change, and capsomeres. (For elaboration, see Duda et al., 1995a; Duda et al., 1995b; Xie and Hendrix, 1995; Conway et al., 1995; Duda, 1998; Hendrix and Duda, 1998. Another keyword is collaboration. We are collaborating with Alasdair Steven and his colleagues James Conway, Naiqian Cheng, and Ramani Lata in the Laboratory of Structural Biology at the NIH, in cryo-electron microscopy studies of the several forms of HK97 capsids. More pictures can be found on our collaborators" web site. We are also collaborating with Jack Johnson and Bill Wikoff at the Scripps Research Institute to determine HK97 capsid structures by X-ray crystallography. The structure of the mature capsid shell is in the final stages of completion, and more structures are ahead. See our collaborators' web site for additional pictures and information. We have collaborated with Dr. John Rosenberg to study complexes formed between the unfolded HK97 capsid protein and the folding chaperone GroEL. These complexes are stable enough to form crystals, and Dr. Rosenberg and his group are well on the way to an atomic resolution structure of these chaperones, caught in the act of carrying out their protein folding role.
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