Summary Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP-2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA. The dsRNA is sheathed within a network of protein domains that include a conserved “helicase” domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of super family 2 of helicases and this structure reveals complex interplay between motor domains, accessory mechanical domains and RNA that has implications for understanding the nanomechanical function this protein family and other ATPases more broadly.

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Defining the functional determinants for RNA surveillance by RIG-IThis study shows that HEL2i-domain mediated scanning allows RIG-I to sense the length of RNA targets, and a short RNA duplex that binds one RIG-I molecule can stimulate ATPase activity and interferon response.

Summary Retinoic acid inducible gene-I (RIG-I) is a key intracellular immune receptor for pathogenic RNAs, particularly from RNA viruses. Here we report the crystal structure of human RIG-I bound to a 5′tri-phosphorylated RNA hairpin and ADP nucleotide at 2.8Å resolution. The RNA ligand contains all structural features that are essential for optimal recognition by RIG-I, as it mimics the panhandle-like signatures within the genome of negative stranded RNA viruses. RIG-I adopts an intermediate, semi-closed conformation in this product state of ATP hydrolysis. The structure of this complex allows us to visualize the first steps in RIG-I recognition and activation upon viral infection.

RNA is a versatile macromolecule that accommodates functional information in primary sequence, secondary and tertiary structure. We use a combination of chemical probing, RNA structure modeling, comparative sequence analysis and functional assays to examine the role of RNA structure in the hepatitis C virus (HCV) genome. We describe a set of conserved but functionally diverse structural RNA motifs that occur in multiple coding regions of the HCV genome, and we demonstrate that conformational changes in these motifs influence specific stages in the virus’s life cycle. Our study shows that these types of structures can pervade a genome, where they play specific mechanistic and regulatory roles, constituting a “code within the code” for controlling biological processes.

Single cell and spatial technologies that profile gene expression across a whole tissue are revolutionizing the resolution of molecular states in clinical tissue samples. Commercially available methods that characterize either single cell or spatial gene expression are currently limited by low sample throughput and/or gene plexy, lack of on-instrument analysis, and the destruction of histological features and epitopes during the workflow. Here, we analyzed large, serial formalin-fixed, paraffin-embedded (FFPE) human breast cancer sections using a novel FFPE-compatible single cell gene expression workflow (Chromium Fixed RNA Profiling; scFFPE-seq), spatial transcriptomics (Visium CytAssist), and automated microscopy-based in situ technology using a 313-plex gene panel (Xenium In Situ). Whole transcriptome profiling of the FFPE tissue using scFFPE-seq and Visium facilitated the identification of 17 different cell types. Xenium allowed us to spatially resolve these cell types and their gene expression profiles with single cell resolution. Due to the non-destructive nature of the Xenium workflow, we were able to perform H&E staining and immunofluorescence on the same section post-processing which allowed us to spatially register protein, histological, and RNA data together into a single image. Integration of data from Chromium scFFPE-seq, Visium, and Xenium across serial sections allowed us to do extensive benchmarking of sensitivity and specificity between the technologies. Furthermore, data integration inspired the interrogation of three molecularly distinct tumor subtypes (low-grade and high-grade ductal carcinoma in situ (DCIS), and invasive carcinoma). We used Xenium to characterize the cellular composition and differentially expressed genes within these subtypes. This analysis allowed us to draw biological insights about DCIS progression to infiltrating carcinoma, as the myoepithelial layer degrades and tumor cells invade the surrounding stroma. Xenium also allowed us to further predict the hormone receptor status of tumor subtypes, including a small 0.1 mm2 DCIS region that was triple positive for ESR1 (estrogen receptor), PGR (progesterone receptor) and ERBB2 (human epidermal growth factor receptor 2, a.k.a. HER2) RNA. In order to derive whole transcriptome information about these cells, we used Xenium data to interpolate the cell composition of Visium spots, and leveraged Visium whole transcriptome information to discover new biomarkers of breast tumor subtypes. We demonstrate that scFFPE-seq, Visium, and Xenium independently provide information about molecular signatures relevant to understanding cancer heterogeneity. However, it is the integration of these technologies that leads to even deeper insights, ushering in discoveries that will progress oncology research and the development of diagnostics and therapeutics.

All canonical transfer RNAs (tRNAs) have a uridine at position 8, involved in maintaining tRNA tertiary structure. However, the hyperthermophilic archaeon Methanopyrus kandleri harbors 30 (out of 34) tRNA genes with cytidine at position 8. Here, we demonstrate C-to-U editing at this location in the tRNA’s tertiary core, and present the crystal structure of a tRNA-specific cytidine deaminase, CDAT8, which has the cytidine deaminase domain linked to a tRNA-binding THUMP domain. CDAT8 is specific for C deamination at position 8, requires only the acceptor stem hairpin for activity, and belongs to a unique family within the “cytidine deaminase–like” superfamily. The presence of this C-to-U editing enzyme guarantees the proper folding and functionality of all M. kandleri tRNAs.

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly. Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus classified within the genus Hepacivirus in the familyFlaviviridae (21). The nonsegmented HCV genome is 9.6-kb and encodes a long polyprotein that is cleaved by cellular and viral proteases into at least 10 distinct products (47,54). The N-terminal region of this polyprotein encodes the viral structural proteins: core and two envelope glycoproteins, E1 and E2. The remainder of the polyprotein encodes nonstructural (NS) proteins: p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Together, the NS proteins regulate intracellular aspects of the viral life cycle, including RNA replication, virus assembly, and viral subversion of host antiviral responses.NS3-4A is an essential enzyme complex of the viral replication machinery and a major target for HCV-specific drug development. The N-terminal domain of NS3 encodes a chymotrypsin-like serine protease that requires NS4A for full activity and is responsible for the cleavage of the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions (47). In addition, the NS3-4A serine protease antagonizes host innate antiviral responses by cleaving the cellular messenger IPS-1 (also known as MAVS, Cardif, and VISA) (43,44,49,53). The C-terminal region of NS3 encodes a superfamily 2 helicase/NTPase domain, which is essential for virus genome replication (36, 41). The serine protease and RNA helicase activities are coordinated, such that the helicase domain can influence the protease activity, and the protease domain can influence RNA helicase activity (7,8,24,40). More recently, a number of genetic studies have implicated the NS3 helicase domain in virus partic...