Population-scale tissue transcriptomics maps long non-coding RNAs to complex disease.

Long non-coding RNA (lncRNA) genes have well-established and important impacts on molecular and cellular functions. However, among the thousands of lncRNA genes, it is still a major challenge to identify the subset with disease or trait relevance. To systematically characterize these lncRNA genes, we used Genotype Tissue Expression (GTEx) project v8 genetic and multi-tissue transcriptomic data to profile the expression, genetic regulation, cellular contexts, and trait associations of 14,100 lncRNA genes across 49 tissues for 101 distinct complex genetic traits. Using these approaches, we identified 1,432 lncRNA gene-trait associations, 800 of which were not explained by stronger effects of neighboring protein-coding genes. This included associations between lncRNA quantitative trait loci and inflammatory bowel disease, type 1 and type 2 diabetes, and coronary artery disease, as well as rare variant associations to body mass index.

The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-γ locus.

Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theiler's [cleanup Salmonella not Theiler's]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-γ (IFN-γ) abundance in activated CD8(+) T cells, increased Theiler's virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.

Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.

IMPORTANCE: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

The Picornaviridae Family: Knowledge Gaps, Animal Models, Countermeasures, and Prototype Pathogens.

Picornaviruses are nonenveloped particles with a single-stranded RNA genome of positive polarity. This virus family includes poliovirus, hepatitis A virus, rhinoviruses, and Coxsackieviruses. Picornaviruses are common human pathogens, and infection can result in a spectrum of serious illnesses, including acute flaccid myelitis, severe respiratory complications, and hand-foot-mouth disease. Despite research on poliovirus establishing many fundamental principles of RNA virus biology and the first transgenic animal model of disease for infection by a human virus, picornaviruses are understudied. Existing knowledge gaps include, identification of molecules required for virus entry, understanding cellular and humoral immune responses elicited during virus infection, and establishment of immune-competent animal models of virus pathogenesis. Such knowledge is necessary for development of pan-picornavirus countermeasures. Defining enterovirus A71 and D68, human rhinovirus C, and echoviruses 29 as prototype pathogens of this virus family may provide insight into picornavirus biology needed to establish public health strategies necessary for pandemic preparedness.

Full-length three-dimensional structure of the influenza A virus M1 protein and its organization into a matrix layer.

Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein-protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy (cryo-EM). The resulting 3.4-Å-resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses.

Differential and convergent utilization of autophagy components by positive-strand RNA viruses.

Many viruses interface with the autophagy pathway, a highly conserved process for recycling cellular components. For three viral infections in which autophagy constituents are proviral (poliovirus, dengue, and Zika), we developed a panel of knockouts (KOs) of autophagy-related genes to test which components of the canonical pathway are utilized. We discovered that each virus uses a distinct set of initiation components; however, all three viruses utilize autophagy-related gene 9 (ATG9), a lipid scavenging protein, and LC3 (light-chain 3), which is involved in membrane curvature. These results show that viruses use noncanonical routes for membrane sculpting and LC3 recruitment. By measuring viral RNA abundance, we also found that poliovirus utilizes these autophagy components for intracellular growth, while dengue and Zika virus only use autophagy components for post-RNA replication processes. Comparing how RNA viruses manipulate the autophagy pathway reveals new noncanonical autophagy routes, explains the exacerbation of disease by starvation, and uncovers common targets for antiviral drugs.

Targeting intramolecular proteinase NS2B/3 cleavages for trans-dominant inhibition of dengue virus.

Many positive-strand RNA viruses translate their genomes as single polyproteins that are processed by host and viral proteinases to generate all viral protein products. Among these is dengue virus, which encodes the serine proteinase NS2B/3 responsible for seven different cleavages in the polyprotein. NS2B/3 has been the subject of many directed screens to find chemical inhibitors, of which the compound ARDP0006 is among the most effective at inhibiting viral growth. We show that at least three cleavages in the dengue polyprotein are exclusively intramolecular. By definition, such a cis-acting defect cannot be rescued in trans This creates the possibility that a drug-susceptible or inhibited proteinase can be genetically dominant, inhibiting the outgrowth of drug-resistant virus via precursor accumulation. Indeed, an NS3-G459L variant that is incapable of cleavage at the internal NS3 junction dominantly inhibited negative-strand RNA synthesis of wild-type virus present in the same cell. This internal NS3 cleavage site is the junction most inhibited by ARDP0006, making it likely that the accumulation of toxic precursors, not inhibition of proteolytic activity per se, explains the antiviral efficacy of this compound in restraining viral growth. We argue that intramolecularly cleaving proteinases are promising drug targets for viruses that encode polyproteins. The most effective inhibitors will specifically target cleavage sites required for processing precursors that exert trans-dominant inhibition.

Exploiting Genetic Interference for Antiviral Therapy.

Rapidly evolving viruses are a major threat to human health. Such viruses are often highly pathogenic (e.g., influenza virus, HIV, Ebola virus) and routinely circumvent therapeutic intervention through mutational escape. Error-prone genome replication generates heterogeneous viral populations that rapidly adapt to new selection pressures, leading to resistance that emerges with treatment. However, population heterogeneity bears a cost: when multiple viral variants replicate within a cell, they can potentially interfere with each other, lowering viral fitness. This genetic interference can be exploited for antiviral strategies, either by taking advantage of a virus's inherent genetic diversity or through generating de novo interference by engineering a competing genome. Here, we discuss two such antiviral strategies, dominant drug targeting and therapeutic interfering particles. Both strategies harness the power of genetic interference to surmount two particularly vexing obstacles-the evolution of drug resistance and targeting therapy to high-risk populations-both of which impede treatment in resource-poor settings.

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites.

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.

Nonlytic viral spread enhanced by autophagy components.

The cell-to-cell spread of cytoplasmic constituents such as nonenveloped viruses and aggregated proteins is usually thought to require cell lysis. However, mechanisms of unconventional secretion have been described that bypass the secretory pathway for the extracellular delivery of cytoplasmic molecules. Components of the autophagy pathway, an intracellular recycling process, have been shown to play a role in the unconventional secretion of cytoplasmic signaling proteins. Poliovirus is a lytic virus, although a few examples of apparently nonlytic spread have been documented. Real demonstration of nonlytic spread for poliovirus or any other cytoplasmic constituent thought to exit cells via unconventional secretion requires demonstration that a small amount of cell lysis in the cellular population is not responsible for the release of cytosolic material. Here, we use quantitative time-lapse microscopy to show the spread of infectious cytoplasmic material between cells in the absence of lysis. siRNA-mediated depletion of autophagy protein LC3 reduced nonlytic intercellular viral transfer. Conversely, pharmacological stimulation of the autophagy pathway caused more rapid viral spread in tissue culture and greater pathogenicity in mice. Thus, the unconventional secretion of infectious material in the absence of cell lysis is enabled by components of the autophagy pathway. It is likely that other nonenveloped viruses also use this pathway for nonlytic intercellular spread to affect pathogenesis in infected hosts.

Double-membraned liposomes sculpted by poliovirus 3AB protein.

Infection with many positive-strand RNA viruses dramatically remodels cellular membranes, resulting in the accumulation of double-membraned vesicles that resemble cellular autophagosomes. In this study, a single protein encoded by poliovirus, 3AB, is shown to be sufficient to induce the formation of double-membraned liposomes via the invagination of single-membraned liposomes. Poliovirus 3AB is a 109-amino acid protein with a natively unstructured N-terminal domain. HeLa cells transduced with 3AB protein displayed intracellular membrane disruption; specifically, the formation of cytoplasmic invaginations. The ability of a single viral protein to produce structures of similar topology to cellular autophagosomes should facilitate the understanding of both cellular and viral mechanisms for membrane remodeling.

Inhibition of cellular autophagy deranges dengue virion maturation.

Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.

Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses.

The high error rates of viral RNA-dependent RNA polymerases create heterogeneous viral populations whose disparate RNA genomes affect each other's survival. We systematically screened the poliovirus genome and identified four sets of dominant mutations. Mutated alleles in capsid- and polymerase-coding regions resulted in dominant negative phenotypes, probably due to the proteins' oligomeric properties. We also identified dominant mutations in an RNA element required for priming RNA synthesis (CRE) and in the protein primer (VPg), suggesting that nonproductive priming intermediates are inhibitory. Mutations that inhibit the activity of viral proteinase 2A were dominant, arguing that inhibition of its known intramolecular activity creates a toxic product. Viral products that, when defective, dominantly interfere with growth of nondefective viruses will probably be excellent drug targets because drug-sensitive viruses should be dominant over drug-resistant variants. Accordingly, a virus sensitive to anticapsid compound WIN51711 dominantly inhibited the intracellular growth of a drug-resistant virus. Therefore, dominant inhibitor screening should validate or predict targets for antiviral therapy with reduced risk for drug resistance.

Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting.

Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA(Lys) uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron-sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans.

Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport.

OBJECTIVE: The lesions of Parkinson disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport. METHODS: We grew primary cortical mouse neurons in microfluidic devices to separate somata from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons. RESULTS: Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts. INTERPRETATION: These results support the hypothesis that the progression of Parkinson disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be trans-synaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extracellular phase of their journey.

Mechanosensitive extrusion of Enterovirus A71-infected cells from colonic organoids.

Enterovirus A71 causes severe disease upon systemic infection, sometimes leading to life-threatening neurological dysfunction. However, in most cases infection is asymptomatic and limited to the gastrointestinal tract, where virus is amplified for transmission. Picornaviruses have previously been shown to exit infected cells via either cell lysis or secretion of vesicles. Here we report that entire Enterovirus A71-infected cells are specifically extruded from the apical surface of differentiated human colon organoids, as observed by confocal microscopy. Differential sensitivity to chemical and peptide inhibitors demonstrated that extrusion of virus-infected cells is dependent on force sensing via mechanosensitive ion channels rather than apoptotic cell death. When isolated and used as inoculum, intact virus-containing extruded cells can initiate new infections. In contrast, when mechanical force sensing is inhibited, large amounts of free virus are released. Thus, extrusion of live, virus-infected cells from intact epithelial tissue is likely to benefit both the integrity of host tissues and the protected spread of this faecal-oral pathogen within and between hosts.

Variant enterovirus A71 found in immune-suppressed patient binds to heparan sulfate and exhibits neurotropism in B-cell-depleted mice.

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease outbreaks with neurological complications and deaths. We previously isolated an EV-A71 variant in the stool, cerebrospinal fluid, and blood of an immunocompromised patient who had a leucine-to-arginine substitution on the VP1 capsid protein, resulting in increased heparin sulfate binding. We show here that this mutation increases the virus's pathogenicity in orally infected mice with depleted B cells, which mimics the patient's immune status, and increases susceptibility to neutralizing antibodies. However, a double mutant with even greater heparin sulfate affinity is not pathogenic, suggesting that increased heparin sulfate affinity may trap virions in peripheral tissues and reduce neurovirulence. This research sheds light on the increased pathogenicity of variant with heparin sulfate (HS)-binding ability in individuals with decreased B cell immunity.

Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays.

Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase-polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.

Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems.

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.