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The Giovanni Armenise-Harvard Foundation Session 2: Membrane Traffic/Macromolecular Entry Overview Each cell is a miniature shipping terminal, busy around the clock as some materials enter and others go out. In endocytosis, essential materials come into the cell through an invagination in the plasma membrane; they are then packaged in vesicles and shuttled to their proper intracellular destinations. The trouble is that these normal endocytic traffic patterns can also be usurped by pathogens, observed Dr. Stephen Harrison, a professor of biochemistry and molecular biology at Harvard. Disease-producing organisms are able to accomplish this because they have evolved ways to breach the cell, escape detection, and make their way to the cytoplasm or the nucleus so they can carry out their harmful business. The speakers in this session discussed how pathogens solve some of those problems. The first two considered the challenges faced by viruses and bacteria that must get past the cell’s plasma membrane without having membranes of their own to fuse with it. The third paper zeroes in on the details of endocytic traffic, and the final talk considers how host cells react to invasion.
Springing the trap - conformational changes associated with poliovirus
cell entry Although poliovirus is an extremely well-studied pathogen, a persistent mystery has been how this uncoated virus enters target cells to replicate. Somehow the virus remains stable in stressful environments (such as the highly acid stomach), is stubbornly inert in the face of non-target cells, yet is poised to fuse with the membrane of the right cell. The secret appears to be a maturation cleavage of a capsid protein precursor (VP0), which occurs in the final stages of virion assembly and locks the virus into a metastable state. This stability enables the virus to survive in the extracellular environment as it travels from cell to cell or host to host. When it encounters the right receptor, poliovirus turns itself inside out, revealing VP4 and the amino terminus of VP1, the two proteins that enable it to attach to membrane. In Dr. Hogle’s lab, this transition—as well as a subsequent transition in which RNA is released from the altered particle—were induced in the absence of receptor by gently heating the virus. The idea of a metastable state that can be reversed by contact with the right receptor grew out of structural and genetic studies of the virus, structural studies of assembly and cell entry intermediates, and thermodynamic and kinetic studies of the in vitro conversions. This model parallels emerging ideas about the maturation and cell entry of more complex enveloped viruses such as influenza and HIV.
Helicobacter pylori virulence factors in the pathogenesis of gastric
diseases The ulcer-causing organism Helicobacter pylori exploits a normal endocytic pathway to enter to the cells of the gut. Once inside, a virulence factor called VacA throws the cell’s normal membrane traffic patterns into chaos. This toxin is activated by low pH and can insert itself into the lipid bilayer. It forms vacuoles that crowd the cytosol and draws large quantities of urease into the cell, causing gridlock that makes it impossible for the cell to carry out its normal functions. Vacuolated cells recycle transferrin normally but are defective in degradation of external ligands such as EGF and in processing of cathepsin precursors, which are secreted in the medium. This may damage the stomach mucosa because acid hydrolases are particularly active in this environment. VacA also inhibits antigen processing, thereby depressing the response of T cells that would ordinarily detect H. pylori antigens on the surface of infected cells and destroy them. VacA also recruits inflammatory cells that erode the mucosal layer. These effects help the pathogen evade destruction so that it can establish a chronic infection. H. pylori is not only extremely well adapted to its niche, but also capable of modifying the environment so that the stomach becomes even more hospitable to the organism. In the future, scientists may be able to use H. pylori to learn more about the physiology of the gut itself.
Protein interactions in clathrin-coated vesicles HIV and some other viruses sneak into the cell via a doorway that is ordinarily used to bring in key nutrients and other essential proteins. By understanding more about the molecular mechanisms that move membrane proteins within eukaryotic cells, Dr. Kirchhausen and his colleagues hope to pave the way for new antiviral treatments. Clathrin coated pits and coated vesicles, formed from clathrin and associated "adaptor" proteins, recruit proteins from the plasma membrane to the endosomal compartment and also convey proteins from the trans-Golgi network (TGN) to the endosome. Entry of important nutrients (e.g., iron and cholesterol), down-regulation of growth-factor receptors, intracellular transport of MHC molecules, membrane recycling, and delivery of degradative enzymes to lysosomes all rely on clathrin-dependent pathways. The clathrin adaptor complex is a four-part structure that recognizes two basic motifs. When it encounters one of these signals, the clathrin complex sorts proteins into vesicles for transport within the cell. Dr. Kirchhausen’s lab has found that this complex recognizes a peptide on the cytosolic tail of CD4, the coreceptor for HIV, and a motif found in the nef protein made by a major HIV virulence gene. In the presence of both CD4 and nef, the virus rapidly enters the cell via the clathrin coated pit. Once the cell is infected, experiments indicate that HIV with a functioning nef gene are able to downregulate the number of CD4 receptors on the cell surface. This may make it easier for formed virions to escape its orbit and proceed to infect other cells. Current investigations are focused on the high resolution X-ray structure of the interface between clathrin and associated adaptor proteins.
Host genetic factors affecting responses to polyoma virus There are two sides to every infection, and although a great deal of research focuses on the pathogen it is equally important to understand what happens in the host. When Dr. Benjamin and his colleagues inoculated newborn mice from more than thirty inbred strains with polyoma viruses known to cause widespread solid tumors, they found a tremendous variation in susceptibility and resistance to infection. One of the polyoma strains they tested is so virulent that it typically kills newborn mice before they have time to develop tumors; other strains produce tumors throughout the body. The researchers quickly saw that different mice responded differently to infection: a virus that produced tumors in 100% of one mouse strain might cause cancer in only 2% of another. Some of the mice resisted even the most virulent of the viruses. The researchers discerned two patterns of susceptibility - one seen only in mice carrying the H2k haplotype and dependent on endogenous superantigen, and the other found in mice with different MHC types and apparently independent of superantigen activity. Mice with the dominant superantigen gene seem to eliminate T cells that might control tumor growth. Two distinct types of resistance have also been noted. Mice with immunologically based resistance develop tumors, instead of dying immediately, when exposed to the most virulent strains of polyoma. Animals with non-immunological resistance are able to block the spread of the virus from the inoculation site.
Symposium Topics
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