RNA Origami: Packaging a Segmented Genome in Orbivirus Assembly and Replication

Understanding how viruses with multi-segmented genomes incorporate one copy of each segment into their capsids remains an intriguing question. Here, we review our recent progress and describe the advancements made in understanding the genome packaging mechanism of a model nonenveloped virus, Bluetongue virus (BTV), with a 10-segment (S1–S10) double-strand RNA (dsRNA) genome. BTV (multiple serotypes), a member of the Orbivirus genus in the Reoviridae family, is a notable pathogen for livestock and is responsible for significant economic losses worldwide. This has enabled the creation of an extensive set of reagents and assays, including reverse genetics, cell-free RNA packaging, and bespoke bioinformatics approaches, which can be directed to address the packaging question. Our studies have shown that (i) UTRs enable the conformation of each segment necessary for the next level of RNA–RNA interaction; (ii) a specific order of intersegment interactions leads to a complex RNA network containing all the active components in sorting and packaging; (iii) networked segments are recruited into nascent assembling capsids; and (iv) select capsid proteins might be involved in the packaging process. The key features of genome packaging mechanisms for BTV and related dsRNA viruses are novel and open up new avenues of potential intervention.     

Structural basis of UGUA recognition by the Nudix protein CFIm25 and implications for a regulatory role in mRNA 3′ processing

Human Cleavage Factor Im (CFI_m) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI_m25) of the CFI_m complex possesses a characteristic α/β/α Nudix fold, CFI_m25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI_m25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI_m25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson–Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap₄A (diadenosine tetraphosphate) by CFI_m25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing.     

Human NLRP3 inflammasome senses multiple types of bacterial RNAs

Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.The innate immune system is the first line of defense against microbial infections. Germ-line–encoded pattern-recognition receptors (PRRs) of the innate immune system recognize the presence of invariant evolutionarily conserved microbial components called “pathogen-associated molecular patterns” (1–3). In response to microbial infections, PRRs rapidly initiate signal-transduction pathways to induce type 1 IFN production, proinflammatory cytokine production, and inflammasome activation. The inflammasome is a cytosolic large caspase-1–containing multiprotein complex that enables autocatalytic activation of caspase-1. Once caspase-1 is activated, it starts to cleave prointerleukin-1β (pro–IL-1β) and prointerleukin-18 (pro–IL-18) proteolytically into bioactive IL-1β and IL-18 (4–7). The mature forms of IL-1β and IL-18 play roles in a variety of infectious and inflammatory processes.Cytosolic microbial nucleic acids are important activators of the innate immune system against both bacterial and viral infections, which induce type 1-IFN and proinflammatory cytokine responses as well as inflammasome activation. The role of microbial nucleic acids in inflammasome activation has been studied mostly in murine bone marrow-derived dendritic cells (BMDCs) or bone marrow-derived macrophages (BMDMs). AIM2 has been identified as a specific cytosolic dsDNA sensor that directly binds ASC (apoptosis-associated speck-like protein containing a carboxyl-terminal CARD-like domain) and forms inflammasome complexes in human and murine cells (8–11).Viral dsRNA was found to activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in human and murine cells (12–15). Several groups have reported that cytosolic bacterial RNA activate the Nlrp3 inflammasome in murine macrophages (13, 16, 17). Our group also has reported that human THP-1–derived macrophages recognize cytosolic bacterial RNA and induce NLRP3 inflammasome activation (12). Bacterial RNA is composed of mRNA, tRNA, and three different sizes of rRNA (23s, 16s, and 5s). Sander et al. (18) reported that, of the different types of Escherichia coli RNA, only E. coli mRNA induced the secretion of IL-1β by murine BMDMs, but E. coli tRNA and E. coli rRNAs did not.We aimed to study (i) whether a variety of cytosolic bacterial RNAs could activate the inflammasome in human myeloid cells and (ii) what types of bacterial RNA activate the inflammasome in human and murine myeloid cells. Here, we demonstrate that a broad spectrum of cytosolic bacterial RNAs strongly induce the cleavage of caspase-1 and the secretion of IL-1β and IL-18 in human macrophages. Human macrophages can sense mRNA, tRNA, rRNAs, and small synthetic ssRNA through NLRP3, but murine macrophages can sense only the mRNA component. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable, but small fragments of bacterial RNA were sufficient to activate the inflammasome. These findings suggest that upon bacterial infections the human and murine NLRP3 inflammasomes sense cytosolic bacterial RNAs differently.     

Positive regulation by small RNAs and the role of Hfq

Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5′ leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an (AAN)₄ Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37 °C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA•mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA•mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.     

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1–3 and 7–8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4–6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.     

An Importin-β-like Protein from Nicotiana benthamiana Interacts with the RNA Silencing Suppressor P1b of the Cucumber Vein Yellowing Virus,Modulating Its Activity

During a plant viral infection, host–pathogen interactions are critical for successful replication and propagation of the virus through the plant. RNA silencing suppressors (RSSs) are key players of this interplay, and they often interact with different host proteins, developing multiple functions. In the Potyviridae family, viruses produce two main RSSs, HCPro and type B P1 proteins. We focused our efforts on the less known P1b of cucumber vein yellowing virus (CVYV), a type B P1 protein, to try to identify possible factors that could play a relevant role during viral infection. We used a chimeric expression system based on plum pox virus (PPV) encoding a tagged CVYV P1b in place of the canonical HCPro. We used that tag to purify P1b in Nicotiana-benthamiana-infected plants and identified by mass spectrometry an importin-β-like protein similar to importin 7 of Arabidopsis thaliana. We further confirmed the interaction by bimolecular fluorescence complementation assays and defined its nuclear localization in the cell. Further analyses showed a possible role of this N. benthamiana homolog of Importin 7 as a modulator of the RNA silencing suppression activity of P1b.     

RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA

SgrS is an Hfq-binding small RNA that is induced under glucose phosphate stress in Escherichia coli. It forms a specific ribo nucleo protein complex with Hfq and RNase E resulting in translational repression and rapid degradation of ptsG mRNA, encoding the glucose transporter. Here, we report translational silencing of ptsG mRNA in a defined in vitro system. We demonstrate that SgrS and Hfq are the minimum components for translational silencing to faithfully reproduce the reaction in cells. We show that ptsG-SgrS base pairing is sufficient to cause translational repression when the ptsG mRNA is forced to base pair with SgrS without the help of Hfq. The extent of translational repression correlates with the extent of duplex formation. We conclude that base pairing itself but not Hfq is directly responsible for translational silencing and the major role of Hfq in gene silencing is to stimulate the base pairing between SgrS and ptsG mRNA. This simple mechanism is in striking contrast to miRNA action in eukaryote in which the RNA is believed to act only as a guide of protein partners.