Cellular Entry of Influenza by Single-particle Imaging

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DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the molecular mechanisms and cellular pathways of influenza infection. To accomplish this goal, we will develop state-of-the-art instrumentation for real-time fluorescence imaging of single viruses and single viral genes. These single-particle approaches will allow us to directly visualize the cellular entry process of influenza viruses in real time, to dissect individual stages of the entry pathway(s) that may be difficult if not impossible to detect by ensemble methods, and to obtain a better understanding of the cellular entry mechanisms of influenza. Influenza, representative of many medically important viruses, is a paradigm for understanding viral entry processes. Influenza viruses enter cells via several steps, including (1) receptor-mediated endocytosis, (2) endocytic trafficking of the viruses to late endosomes where viral fusion with endosomes leads to the release viral genes into the cytoplasm, and (3) nuclear import of these viral genes. Three sets of experiments are designed to investigate these viral entry steps. Specific Aim 1: Single-particle tracking will be used to investigate the endocytosis mechanisms used by the influenza viruses. We will address important questions including: (i) which cellular endocytic pathways are exploited by influenza to enter cells; (ii) what are the molecular characteristics of these pathways; (iii) How viruses are targeted to endocytic machinery; and/or (iv) how endocytic machinery assembles around viruses? Specific Aim 2: Single-particle tracking will be used to elucidate the endocytic trafficking of influenza viruses inside cells. We will address: (i) how viruses are transported inside cells; (ii) how viruses are trafficked between different endocytic compartments; and (iii) how viruses are sorted. Specific Aim 3: Single-particle tracking will be used to characterize the regulated nuclear import of influenzal genes, in the form of viral ribonucleoproteins (vRNPs). We will address: (i) the transport mechanisms of vRNPs in cells and (ii) the molecular mechanisms that regulate the nuclear import of vRNPs? The above experiments promise to provide new insights into the cellular entry process of influenza viruses. The new methodology developed here can be extended to study other families of viruses and may in the future lead to new designs of anti-viral drugs and therapeutics. These techniques can also be applied to other molecular and cellular biological systems.