ISG15/USP18/STAT2 is a molecular hub regulating IFN I-mediated control of Dengue and Zika virus replication.

The establishment of a virus infection is the result of the pathogen's ability to replicate in a hostile environment generated by the host's immune system. Here, we found that ISG15 restricts Dengue and Zika viruses' replication through the stabilization of its binding partner USP18. ISG15 expression was necessary to control DV replication driven by both autocrine and paracrine type one interferon (IFN-I) signaling. Moreover, USP18 competes with NS5-mediated STAT2 degradation, a major mechanism for establishment of flavivirus infection. Strikingly, reconstitution of USP18 in ISG15-deficient cells was sufficient to restore the STAT2's stability and restrict virus growth, suggesting that the IFNAR-mediated ISG15 activity is also antiviral. Our results add a novel layer of complexity in the virus/host interaction interface and suggest that NS5 has a narrow window of opportunity to degrade STAT2, therefore suppressing host's IFN-I mediated response and promoting virus replication.

Myeloid cell interferon secretion restricts Zika flavivirus infection of developing and malignant human neural progenitor cells.

Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.

The flavivirus polymerase NS5 regulates translation of viral genomic RNA.

Flaviviruses, including dengue virus and Zika virus, contain a single-stranded positive sense RNA genome that encodes viral proteins essential for replication and also serves as the template for new genome synthesis. As these processes move in opposite directions along the genome, translation must be inhibited at a defined point following infection to clear the template of ribosomes to allow efficient replication. Here, we demonstrate in vitro and in cell-based assays that the viral RNA polymerase, NS5, inhibits translation of the viral genome. By reconstituting translation in vitro using highly purified components, we show that this translation block occurs at the initiation stage and that translation inhibition depends on NS5-RNA interaction, primarily through association with the 5' replication promoter region. This work supports a model whereby expression of a viral protein signals successful translation of the infecting genome, prompting a switch to a ribosome depleted replication-competent form.

Circularization of flavivirus genomic RNA inhibits de novo translation initiation.

Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA-RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome.

Rotavirus Genomic RNA Complex Forms via Specific RNA-RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity.

Rotavirus (RV), a member of the Reoviridae family, causes infection in children and infants, with high morbidity and mortality. To be viable, the virus particle must package a set of eleven RNA segments. In order to understand the packaging mechanism, here, we co-synthesized sets of RNA segments in vitro in different combinations and detected by two alternate methods: the electrophoretic mobility shift assay (EMSA) and the RNA-bead pull-down assay. We showed that viral positive-sense RNA segments interact with each other in a specific manner, forming RNA complexes, and that the RNA-RNA interactions followed a sequential order initiated by small RV segments. Further, we demonstrated that RNA complexes were perturbed by targeted specific antisense oligoribonucleotides (ORNs) complementary to short RNA sequences, indicating that the RNA-RNA interactions between different segments were sequence-specific. The same inhibitory ORNs also had the capability to inhibit virus replication. The combined in vitro and in vivo data inferred that RNA-RNA interactions and specific complex formation are essential for sorting different segments, possibly prior to, or during, genome packaging. As genome assembly is a universal requirement in the Reoviridae family, this work offers an approach towards a further understanding of the sorting and packaging mechanisms of RV and related dsRNA (double-stranded RNA) viruses.

Generation of infectious RNA complexes in Orbiviruses: RNA-RNA interactions of genomic segments.

Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of Epizootic Haemorrhagic Disease Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules.

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.

Exploring IRES region accessibility by interference of foot-and-mouth disease virus infectivity.

Translation initiation of picornavirus RNA is driven by an internal ribosome entry site (IRES) element located upstream of the initiator codon. RNA structure organization as well as RNA-protein interaction plays a fundamental role in internal initiation. IRES activity has been mainly analyzed in the context of reporter genes, lacking regions of the viral genome potentially affecting translation efficiency. With the aim to understand the vulnerability of the IRES and translation start region to small molecules in the context of the viral genome, we designed a set of customized RNase-resistant 2'O-methyl antisense oligoribonucleotides (2'OMe AONs) based on RNA structure data. These AONs were then used to monitor their capacity to interfere viral RNA translation, and thus, to inhibit virus yield. Foot-and-mouth disease virus (FMDV) RNA translation can be initiated at two in-frame AUG codons. We show here that a 2'OMe AON complementary to AUG2 inhibited viral multiplication more efficiently than the one that targeted AUG1. Furthermore, the response of the viral RNA to AONs targeting the IRES region denoted important differences between tissue culture cells and cell-free systems, reinforcing the need to analyze viral RNA response in living cells. Importantly, we have identified four specific motifs within the IRES element that are targets for viral inhibitors both in tissue culture cells and in cell-free systems. The identified targets define accessible regions to small molecules, which disturb either the RNA structural organization or the RNA-protein interactions needed to initiate translation in FMDV RNA.