The discovery of regulatory small RNAs continues to reshape paradigms in both molecular biology and virology. Here we describe examples of influenza A virus-derived small viral RNAs (svRNAs). svRNAs are 22-27 nt in length and correspond to the 5′ end of each of the viral genomic RNA (vRNA) segments. Expression of svRNA correlates with the accumulation of vRNA and a bias in RNAdependent RNA polymerase (RdRp) activity from transcription toward genome replication. Synthesis of svRNA requires the RdRp, nucleoprotein and the nuclear export protein NS2. In addition, svRNA is detectable during replication of various influenza A virus subtypes across multiple host species and associates physically with the RdRp. We demonstrate that depletion of svRNA has a minimal impact on mRNA and complementary vRNA (cRNA) but results in a dramatic loss of vRNA in a segment-specific manner. We propose that svRNA triggers the viral switch from transcription to replication through interactions with the viral polymerase machinery. Taken together, the discovery of svRNA redefines the mechanistic switch of influenza virus transcription/replication and provides a potential target for broad-range, anti-influenza virus-based therapeutics.microRNA | replicase | replication switch | RNA dependent RNA polymerase | transcriptase

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.

Middle East respiratory syndrome coronavirus (MERS-CoV) remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH) with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.