The long-term goals of this work are to determine the mechanisms by which components of the autophagy pathway promote viral replication and spread and to identify targets for development of non-toxic antiviral compounds. The canonical autophagy (self-eating) pathway, described by recent Nobel Prize winner Yoshinori Ohsumi, is stimulated by nutrient deprivation and culminates in the degradation of cytoplasmic contents, thus nourishing the starving cell. To this end, dramatic cellular events such as massive lipid scavenging, growth of novel membranous compartments, entrapment of cytoplasm by both concave and convex membrane curvature, and fusion with lysosomes are accomplished within minutes. Numerous microbes, including poliovirus and Dengue virus, have evolved to subvert segments of the cellular autophagy pathway or its individual constituents to promote their infectious cycles. The precise individual contributions of genes and proteins from autophagy pathways have not been identified for any of these viruses. In the experiments proposed here, CRISPR/Cas9 technology will be used to generate matched cell lines oblated for individual steps such as autophagy initiation, expansion and curvature of the limiting membrane, and placement of crucial fusion protein LC3 on the surface of the nascent autophagosome. Testing the effects of individual gene deletions on the entry, translation, RNA replication and morphogenesis of poliovirus and dengue virus will reveal which components of the autophagy pathway are usurped by these two representative positive- strand RNA viruses. The central hypothesis, based on preliminary results, is that poliovirus and dengue virus use distinct steps and components of the cellular autophagy pathway for disparate purposes: RNA replication and nonlytic spread for poliovirus, virion assembly and maturation for dengue virus. The rationale of the proposed research is that, once the components and mechanisms of these subversive events are identified, it will be feasible to target antiviral compounds to particular molecules and processes. Not surprisingly, given the dependence of these viruses on autophagy machinery, short periods of fasting greatly exacerbate pathogenesis in mouse models of both poliovirus and dengue virus infection. This exacerbation is also observed in response to commonly used medications known to stimulate autophagy. Rigorous genetic tests will be used to determine whether this increased pathogenesis is indeed dependent on the cellular autophagy pathway.
The proposed research to investigate the mechanisms by which components of the ancient recycling pathways of autophagy ('self-eating') facilitate RNA viral infections is relevant to public health because it provides new insights into the growth and pathogenesis of burdensome and often deadly viruses. The promotion of viral growth by autophagy pathways and components, once understood, will identify new antiviral targets and suggest new public health measures, such as warnings against commonly used medications that exacerbate viral disease in animal models by promoting cellular autophagy. These insights are relevant to NIH missions of fostering creative, fundamental discoveries and using this new information to prevent and cure human disease.
