Positive-strand RNA viruses that infect eukaryotic cells all rearrange intracellular membranes and replicate their RNA genomes on the topologically cytoplasmic surfaces. Several of these viruses, including poliovirus and Dengue virus, mimic or subvert the cellular pathway of autophagy to form double-membraned intracellular compartments. Experiments are proposed to explore the hypothesis that these membranes facilitate viral exit from infected cells: for poliovirus, because the double-membraned topology can allow non-lytic exit of virus and other cytoplasmic constituents, and for Dengue virus, because the pathway or products of cellular autophagy are required for infectious virion assembly. To investigate the contribution of double-membraned vesicles during poliovirus infection of tissue-culture cells and mice, live-cell time-lapse microscopy, pharmaceutical perturbations, and viral and mouse genetics are employed to distinguish between lytic and non-lytic, and between apoptotic and non-apoptotic spread. These principles are also applied to the study of hepatitis A virus, a picornavirus whose ability to spread through the liver with no apparent cell lysis remains mysterious and may be explained by the topology of the double-membraned vesicles induced during infection. A single poliovirus protein, 3AB, was found to be sufficient to create double-membraned from single-membraned liposomes, and the hypothesis that this mechanism might be shared with cellular autophagy proteins tested. The novel and dramatic dependence of Dengue virus infectivity on a functioning cellular autophagy pathway will be explored mechanistically by determining the biochemical defect in the non-infectious virions produced when cellular autophagy is inhibited. The versatile cellular process of autophagy, although known to be part of the innate immune response, provides a new target for antiviral control.
The process of cellular 'autophagy', literally 'self-eating', was originally discovered as a primitive and ancient cellular response to starvation. However, many microbes, including viruses such as poliovirus and Dengue virus, have evolved not only to avoid, but to mimic and subvert, this ancient cellular process. The hypotheses to be explored in this proposal is that the process of wrapping up macromolecules for degradation is also a way to deliver macromolecules outside infected cells, that this process can be co-opted by infecting viruses to accomplish their spread from cell to cell, and that pharmaceuticals being developed to enhance and inhibit autophagy are a novel class of antiviral compounds.
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