Discovered in 2017, Colletotrichum camelliae filamentous virus 1 (CcFV-1) is considered an outlier among dsRNA viruses due to its filamentous morphology. Typical dsRNA viruses possess a spherically shaped icosahedral capsid with multiple copies of a viral polymerase complex packaged inside for in situ transcription of the viral genome. These dsRNA viruses do not completely uncoat during host entry, allowing transcription of the viral genome to take place within intact capsids while avoiding dsRNA activated defense mechanisms of their hosts. In contrast, CcFV-1 particles assume the shape of a flexuous filament with a 12.2 kb genome that is organized into eight gene segments with ten predicted open reading frames (ORFs) including P1, an RNA- dependent RNA polymerase (RdRP); P3, a putative S-adenosyl methionine (SAM)-dependent methyltransferase; P4, the capsid protein (CP); and seven other proteins of yet unknown functions. The overarching goals of this grant are to determine how CcFV-1 organizes its genomic dsRNA inside the viral particle, whether CcFV-1 has virion-associated transcription activity and, if so, how the filamentous CcFV-1 capsid provides polymerase access to the dsRNA genome without complete uncoating and disassembly. We have already demonstrated that recombinant CcFV-1 CP forms both helical VLPs and small oligomers. Recombinant P1 RdRP forms stable dimers and we have obtained a preliminary cryo-electron microscopy reconstruction of P1 showing a S-shaped molecule. CcFV-1 P1 and P3 form a stable complex that is also suitable for cryo-EM analysis. In addition, we will have access to infectious CcFV-1 virion samples provided by Dr. Wenxing Xu at the Huazhong Agricultural University in China. Altogether, these data not only provide a strong rationale, but also demonstrate the feasibility of our proposed experiments. Our research plan consists of three independent but complementary aims. Aim 1 is to elucidate the structural organization of the CcFV-1 viral particle by solving the structure of both the filamentous VLP and infectious virion. We hypothesize that the helical capsid of CcFV-1 protects its dsRNA genome from triggering cellular antiviral responses during all stages of viral replication. Aim 2 is to determine the structural and biochemical properties of the CcFV-1 polymerase complex. The central hypothesis is that CcFV-1 polymerase complex should possess RdRP, methyltransferase, and guanylyltransferase activities with possibly unique structural features that allow it to transcribe dsRNA packaged in a helical capsid. Aim 3 is explore interactions between the CcFV-1 capsid and the polymerase complex. We will analyze the RNA transcription activity of infectious viral particles and investigate the mechanism of polymerase incorporation into infectious virions. We hypothesize that the CcFV-1 polymerase complex is likely localized to the terminal ends of the viral helical capsid through direct protein- protein and/or protein-RNA interac...