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Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention. Coronaviruses can infect the nose and throat and are a main cause of the common cold. Infections are usually mild and short-lived, but sometimes they can turn nasty. In 2002 and 2012, two dangerous new coronaviruses emerged and caused diseases known as SARS and MERS. These viruses caused much more serious symptoms and in some cases proved deadly. The question is, why are some coronaviruses more dangerous than others? Scientists know that the body's response to virus infection can make a difference to whether someone had mild or severe disease. So, to understand why some coronaviruses cause a cold and others kill, they also need to learn how people react to virus infection. Coronaviruses hijack membranes inside cells and turn them into virus factories. Within these factories, the viruses build molecular machinery called replicase complexes to copy their genetic code, which is needed for the next generation of virus particles. The viruses steal and repurpose proteins from their host cell that will assist in the copying process. However, scientists do not yet know which host proteins are essential for the virus to multiply. So, to find out, V’kovski et al. developed a way to tag any host protein that came near the virus factories. The new technique involved attaching an enzyme called a biotin ligase to the replicase complex. This enzyme acts as a molecular label gun, attaching a chemical tag to any protein that comes within ten nanometres. The label gun revealed that more than 500 different proteins come into contact with the replicase complex. To find out what these proteins were doing, the next step was to switch off their genes one by one. This revealed the key cell machinery that coronaviruses hijack when they are replicating. It included the cell's cargo transport system, the waste disposal system, and the protein production system. Using these systems allows the viruses to copy their genetic code next to machines that can turn it straight into viral proteins. These new results provide clues about which proteins viruses actually need from their host cells. They also do not just apply to coronaviruses. Other viruses use similar strategies to complete their infection cycle. These findings could help researchers to understand more generally about how viruses multiply. In the future, this knowledge could lead to new ways to combat virus infections.

Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymeraseó (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genomewide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing.

Background Chromatin modifications such as DNA methylation are targeted to transposable elements by small RNAs in a process termed RNA-directed DNA methylation (RdDM). In plants, canonical RdDM functions through RNA polymerase IV to reinforce pre-existing transposable element silencing. Recent investigations have identified a “non-canonical” form of RdDM dependent on RNA polymerase II expression to initiate and re-establish silencing of active transposable elements. This expression-dependent RdDM mechanism functions through RNAi degradation of transposable element mRNAs into small RNAs guided by the RNA-dependent RNA polymerase 6 (RDR6) protein and is therefore referred to as RDR6-RdDM. Results We performed whole-genome MethylC-seq in 20 mutants that distinguish RdDM mechanisms when transposable elements are either transcriptionally silent or active. We identified a new mechanism of expression-dependent RdDM, which functions through DICER-LIKE3 (DCL3) but bypasses the requirement of both RNA polymerase IV and RDR6 (termed DCL3-RdDM). We found that RNA polymerase II expression-dependent forms of RdDM function on over 20 % of transcribed transposable elements, including the majority of full-length elements with all of the domains required for autonomous transposition. Lastly, we find that RDR6-RdDM preferentially targets long transposable elements due to the specificity of primary small RNAs to cleave full-length mRNAs. Conclusions Expression-dependent forms of RdDM function to critically target DNA methylation to full-length and transcriptionally active transposable elements, suggesting that these pathways are key to suppressing mobilization. This targeting specificity is initiated on the mRNA cleavage-level, yet manifested as chromatin-level silencing that in plants is epigenetically inherited from generation to generation.

RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) is a key RNA silencing factor initially characterized in transgene silencing and virus resistance. This enzyme also contributes to the biosynthesis of endogenous short interfering RNAs (siRNAs) from non-coding RNAs, transposable elements and protein-coding transcripts. One class of protein-coding transcripts that have recently emerged as major sources of RDR6-dependent siRNAs are nucleotide-binding leucine-rich repeat (NB-LRR) proteins, a family of immune-receptors that perceive specific pathogen effector proteins and mount Effector-Triggered Immunity (ETI). Nevertheless, the dynamic post-transcriptional control of NB-LRR transcripts during the plant immune response and the functional relevance of NB-LRRs in signaling events triggered by Pathogen-Associated Molecular Patterns (PAMPs) remain elusive. Here, we show that PTI is constitutive and sensitized in the Arabidopsis rdr6 loss-of-function mutant, implicating RDR6 as a novel negative regulator of PTI. Accordingly, rdr6 mutant exhibits enhanced basal resistance towards a virulent Pseudomonas syringae strain. We further provide evidence that dozens of CC-NB-LRRs (CNLs), including the functionally characterized RPS5 gene, are post-transcriptionally controlled by RDR6 both constitutively and during PTI. These CNL transcripts are also regulated by the Arabidopsis microRNA miR472 and knock-down of this miRNA recapitulates the PTI and basal resistance phenotypes observed in the rdr6 mutant background. Furthermore, both miR472 and rdr6 mutants were more resistant to Pto DC3000 expressing AvrPphB, a bacterial effector recognized by the disease resistance protein RPS5, whereas transgenic plants overexpressing miR472 were more susceptible to this bacterial strain. Finally, we show that the enhanced basal and RPS5-mediated resistance phenotypes observed in the rdr6 mutant are dependent on the proper chaperoning of NB-LRR proteins, and might therefore be due to the enhanced accumulation of CNL proteins whose cognate mRNAs are no longer controlled by RDR6-dependent siRNAs. Altogether, this study supports a model whereby the miR472- and RDR6-mediated silencing pathway represents a key regulatory checkpoint modulating both PTI and ETI responses through the post-transcriptional control of disease resistance genes.

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5'-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3' half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culturederived particles (HEVcc) with viral titers between 10⁵ and 10⁶ FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral–host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen

The crystal structure of bat influenza A polymerase bound to a serine-5 phosphorylated peptide mimic from the C-terminal domain of cellular RNA polymerase II shows how the two polymerases are directly coupled and suggests that the interaction site could be targeted for antiviral drug development. Bound FluA polymerase structure determined Influenza virus replication requires a close coupling of viral and cellular transcription so that the influenza virus polymerase can snatch 5′-capped primers from nascent Pol II transcripts for transcription priming. Stephen Cusack and colleagues now present a crystal structure of bat FluA polymerase bound to a Pol II C-terminal domain peptide-mimic. They show how the two polymerases interact and suggest that the interaction site could be targeted for antiviral drug development. The heterotrimeric influenza polymerase (FluPol), comprising subunits PA, PB1 and PB2, binds to the conserved 5′ and 3′ termini (the ‘promoter’) of each of the eight single-stranded viral RNA (vRNA) genome segments and performs both transcription and replication of vRNA in the infected cell nucleus 1 , 2 , 3 . To transcribe viral mRNAs, FluPol associates with cellular RNA polymerase II (Pol II) 4 , 5 , 6 , 7 , which enables it to take 5′-capped primers from nascent Pol II transcripts 8 , 9 . Here we present a co-crystal structure of bat influenza A polymerase bound to a Pol II C-terminal domain (CTD) peptide mimic, which shows two distinct phosphoserine-5 (SeP5)-binding sites in the polymerase PA subunit, accommodating four CTD heptad repeats overall. Mutagenesis of the SeP5-contacting basic residues (PA K289, R454, K635 and R638) weakens CTD repeat binding in vitro without affecting the intrinsic cap-primed (transcription) or unprimed (replication) RNA synthesis activity of recombinant polymerase, whereas in cell-based minigenome assays the same mutations substantially reduce overall polymerase activity. Only recombinant viruses with a single mutation in one of the SeP5-binding sites can be rescued, but these viruses are severely attenuated and genetically unstable. Several previously described mutants that modulate virulence can be rationalized by our results, including a second site mutation (PA(C453R)) that enables the highly attenuated mutant virus (PA(R638A)) to revert to near wild-type infectivity 10 . We conclude that direct binding of FluPol to the SeP5 Pol II CTD is fine-tuned to allow efficient viral transcription and propose that the CTD-binding site on FluPol could be targeted for antiviral drug development.