Cell entry and genome replication are two essential processes of any viral life cycle. The atomic details of these processes are largely unknown for large non-enveloped viruses, unlike enveloped viruses like flu, AIDS and herpes viruses. Upon cell entry, non-enveloped dsRNA viruses sense environmental changes for internal transcription activation. We have studied non-enveloped dsRNA viruses with a single-layered (cytoplasmic polyhedrosis virus - CPV), a double-layered (aquareovirus), and a triple-layered (bluetongue virus ? or BTV ? a member of the Reoviridae family) capsid. Because of its well studied molecular biology and the existence of a reverse genetics system, BTV in particular serves as a good model system for studying cell entry and transcription by such viruses. Thus, the goal of this project is to use state-of-the-art cryo electron microscopy and tomography to determine the structural basis of dsRNA virus cell entry and genome transcription. Our results on BTV show that VP5 contains features similar to membrane fusion proteins and undergoes significant conformational changes at low pH to form a filamentous trimer structure. We hypothesize that this filamentous structure interacts and subsequently breaks endosomal membrane during cell entry. Using the simplest member (CPV) of the Reoviridae, we recently determined organization of dsRNA genome and transcriptional enzyme complex, and showed that RNA transcription activation is mediated by the SAM- dependent ATPase activity of its capping protein. This result, together with earlier observations in CPV and other members of the Reoviridae that RNA transcription activities were coupled with ATP hydrolysis (and more recently with viral ATPase activity), lead to our second hypothesis that the BTV capping protein (VP4) also contains an ATPase, which, triggered by the removal of outer shell, mediates activation of BTV RNA transcription.
In Aim 1, we will incubate BTV virions and recombinant VP5 (wild-type and mutants) with liposomes at neutral and low pH and observe possible molecular interactions between viral particles and lipid membrane with cryo electron tomography (cryoET). Such direct structural data ? expected at nanometer resolution with phase plate and energy-filtering technologies and subtomogram averaging ? will test our first hypothesis and clarify whether and how VP5 penetrates liposomes.
Aims 2 -3 will test our hypothesis on the mechanism of RNA transcription. First, to clarify how VP5 detachment triggers conformational changes, we will determine the atomic structures of the BTV virion (pre-triggering, all transcription substrates) and cores (post- triggering: with only ATP or under transcribing condition) by icosahedral reconstruction (Aim 2). Second, to learn how these conformational changes lead to transcription activation, we will determine the atomic structures of transcriptional enzyme complex and dsRNA genome organization of cores (transcribing, ATP) and virions (all transcription substrates) by asymmetric reconstruction (Aim 3). These studies will be complemented by structure-based mutagenesis with our established method to obtain recombinant particles.
Viruses in the Reoviridae family infect a wide range of hosts, including animals, plants, fungi, and bacteria; within this family, rotaviruses cause 450,000 deaths worldwide each year, and BTV kills livestock, with devastating economic and social consequences. The proposed structural and functional studies focus on BTV to provide structural answers to fundamental questions of cell entry and transcription regulation of these and other dsRNA viruses. The results will provide profound insight into the structural basis of dsRNA virus replication and inform rational development of anti-viral drugs and vaccines against these viruses.
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