Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 Å, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.

Ubiquitin (Ub) signaling plays a key regulatory role in nearly every aspect of eukaryotic biology and is initiated by E1 enzymes that activate and transfer Ub to E2 Ub-conjugating enzymes. Despite Ub E1's fundamental importance to the cell and its attractiveness as a target for therapeutic intervention in cancer and other diseases, its only available structural information is derived from yeast orthologs of human ubiquitin-like modifieractivating enzyme 1 (hUBA1). To illuminate structural differences between yeast and hUBA1 structures that might be exploited for the development of small-molecule therapeutics, we determined the first crystal structure of a hUBA1-Ub complex. Using structural analysis, molecular modeling, and biochemical analysis, we demonstrate that hUBA1 shares a conserved overall structure and mechanism with previously characterized yeast orthologs, but displays subtle structural differences, particularly within the active site. Computational analysis revealed four potential ligand-binding hot spots on the surface of hUBA1 that might serve as targets to inhibit hUBA1 at the level of Ub activation or E2 recruitment or that might potentially be used in approaches such as protein-targeting chimeric molecules. Taken together, our work enhances our understanding of the hUBA1 mechanism, provides an improved framework for the development of small-molecule inhibitors of UBA1, and serves as a stepping stone for structural studies that involve the enzymes of the human Ub system at the level of both E1 and E2.

Renal cell carcinoma (RCC) is known for its multidrug resistance. Using data obtained from the cancer transcriptome database Oncomine and the proteome database The Human Protein Atlas, we identified the repression of organic cation transporter OCT2 as a potential factor contributing to oxaliplatin resistance in RCC. By analyzing OCT2 expression in collected patient tissues and commercial tissue microarray specimens, we demonstrated OCT2 repression in RCC at both transcription and protein levels. Epigenetic analysis revealed that the repressed OCT2 promoter in RCC is characterized by hypermethylated CpG islands and the absence of H3K4 methylation. Further mechanistic studies showed that DNA hypermethylation blocked MYC activation of OCT2 by disrupting its interaction with the E-Box motif, which prevented MYC from recruiting MLL1 to catalyze H3K4me3 at the OCT2 promoter and resulted in repressed OCT2 transcription. Targeting this mechanism, we designed a sequential combination therapy and demonstrated that epigenetic activation of OCT2 by decitabine sensitizes RCC cells to oxaliplatin both in vitro and in xenografts. Our study highlights the potential of translating "omics" data into the development of targeted therapies.