Comprehensive in virio structure probing analysis of the influenza A virus identifies functional RNA structures involved in viral genome replication.

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures of vRNA are known to be involved in the viral proliferation process. Comprehensive vRNA structures in vitro, in virio, and in cellulo have been analyzed. However, the resolution of the structure map can be improved by comparative analysis and statistical modeling. Construction of a more high-resolution and reliable RNA structure map can identify uncharacterized functional structure motifs on vRNA in virion. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-seq with a robust statistical analysis. Our high-resolution analysis identified a stem-loop structure at nucleotide positions 39 - 60 of segment 6 and further validated the structure at nucleotide positions 87 - 130 of segment 5 that was previously predicted to form a pseudoknot structure in silico. Notably, when the cells were infected with recombinant viruses which possess the mutations to disrupt the structure, the replication and packaging of the viral genome were drastically decreased. Our results provide comprehensive and high-resolution information on the influenza A virus genome structures in virion and evidence that the functional RNA structure motifs on the influenza A virus genome are associated with appropriate replication and packaging of the viral genome.

Azo-tagged C4N4 fluorophores: unusual overcrowded structures and their application to fluorescent imaging.

The C4N4 fluorophore is an intense fluorescence emitter featuring a 2,5-diaminopyrimidine core comprising four carbon and four nitrogen atoms. A series of C4N4 derivatives was photochemically dimerized at the 5-amino group, furnishing overcrowded ortho-tetraaryl-substituted diaryl azo compounds with a characteristic skewed structure revealed by X-ray crystallography. The photoquenched azo-C4N4s are useful for fluorescently visualizing cells under hypoxic conditions.

RNA Structure Determination by High-Throughput Structural Analysis.

RNA functions are closely linked with their structures. Therefore, elucidating the secondary structure of RNAs provides crucial information regarding their function. The chemical modification or RNase-mediated digestion of single-stranded RNA has been utilized to experimentally reveal RNA secondary structures. Owing to advances in high-throughput sequencing technology and chemical analysis, RNA structural analyses that enable structural profiling at the transcriptomic scale in living cells have been developed. Here, we provide an overview of the high-throughput RNA structural (HTS) analyses and describe the computational processing steps of recent HTS analysis pipelines: PROBer, BUMHMM, and reactIDR.

A novel E198K substitution in the PA gene of influenza A virus with reduced susceptibility to baloxavir acid.

Baloxavir acid (BXA), the active compound in baloxavir marboxil (BXM), reduces the propagation of influenza A and B viruses by inhibiting the cap-dependent endonuclease activity of the polymerase acidic (PA) subunit. Although BXM has been used to treat influenza virus infections, recently, there has been general concern about the emergence of viruses with low susceptibility to BXA. Here, we identified a novel PA subunit substitution, PA E198K, that reduced susceptibility to BXA. The IC50 of BXA toward influenza A viruses containing PA E198K increased approximately 2- to 6-fold. These findings help to understand the mechanism by which PA substitutions reduce susceptibility to BXA.

Anti-influenza Virus Activity of Methylthio-Formycin Distinct From That of T-705.

Seasonal influenza virus epidemics result in severe illness, and occasionally influenza pandemics cause significant morbidity and mortality, although vaccines and anti-influenza virus drugs are available. By screening an in-house library, we identified methylthio-formycin (SMeFM), an adenosine analog, as a potent inhibitor of influenza virus propagation. SMeFM inhibited the propagation of influenza A and B viruses (IC50: 34.1 and 37.9 nM, respectively) and viruses showing reduced susceptibility to baloxavir and neuraminidase inhibitors but not T-705 (Favipiravir). However, the combination of T-705 and SMeFM inhibited the propagation of the influenza virus not in an antagonistic but in a slightly synergistic manner, suggesting that SMeFM has targets distinct from that of T-705. SMeFM induced A-to-C transversion mutations in virus genome RNA, and SMeFM triphosphate did not inhibit in vitro viral RNA synthesis. Our results show that SMeFM inhibits the propagation of the influenza virus by a mechanism different from that of T-705 and is a potential drug candidate to develop for anti-influenza drug.

Synthesis and antiviral activity of formycin derivatives with anti-influenza virus activity.

In a screening using our unique natural product library, the C-nucleoside antibiotic formycin A, which exerts strong anti-influenza virus activity, was rediscovered. Aiming to develop a new type of anti-influenza virus drug, we synthesized new derivatives of formycin and evaluated its anti-influenza virus activity. Structural modifications were focused on the base moiety and sugar portion, respectively, and >40 novel formycin derivatives were synthesized. Modification of the C-7 position of the pyrazolopyrimidine ring strongly contributed to improve the activity. In particular, excellent anti-influenza virus activity was observed in the NHMe (10), SMe (12), and SeMe (15) derivatives, in which heteroatoms were introduced. In addition, in the modification of the sugar moiety, the presence of a hydroxyl group and its stereochemistry greatly affected both the expression and intensity of the activity. Furthermore, the evaluation results of the 7-SEt derivative (29) and the 2'-modified derivative (59) suggested that structural modifications may reduce cytotoxicity.

Development of a Genetically Stable Live Attenuated Influenza Vaccine Strain Using an Engineered High-Fidelity Viral Polymerase.

RNA viruses demonstrate a vast range of variants, called quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerase. Although live attenuated vaccines are effective in preventing RNA virus infection, there is a risk of reversal to virulence after their administration. To test the hypothesis that high-fidelity viral polymerase reduces the diversity of influenza virus quasispecies, resulting in inhibition of reversal of the attenuated phenotype, we first screened for a high-fidelity viral polymerase using serial virus passages under selection with a guanosine analog ribavirin. Consequently, we identified a Leu66-to-Val single amino acid mutation in polymerase basic protein 1 (PB1). The high-fidelity phenotype of PB1-L66V was confirmed using next-generation sequencing analysis and biochemical assays with the purified influenza viral polymerase. As expected, PB1-L66V showed at least two-times-lower mutation rates and decreased misincorporation rates, compared to the wild type (WT). Therefore, we next generated an attenuated PB1-L66V virus with a temperature-sensitive (ts) phenotype based on FluMist, a live attenuated influenza vaccine (LAIV) that can restrict virus propagation by ts mutations, and examined the genetic stability of the attenuated PB1-L66V virus using serial virus passages. The PB1-L66V mutation prevented reversion of the ts phenotype to the WT phenotype, suggesting that the high-fidelity viral polymerase could contribute to generating an LAIV with high genetic stability, which would not revert to the pathogenic virus.IMPORTANCE The LAIV currently in use is prescribed for actively immunizing individuals aged 2 to 49 years. However, it is not approved for infants and elderly individuals, who actually need it the most, because it might prolong virus propagation and cause an apparent infection in these individuals, due to their weak immune systems. Recently, reversion of the ts phenotype of the LAIV strain currently in use to a pathogenic virus was demonstrated in cultured cells. Thus, the generation of mutations associated with enhanced virulence in LAIV should be considered. In this study, we isolated a novel influenza virus strain with a Leu66-to-Val single amino acid mutation in PB1 that displayed a significantly higher fidelity than the WT. We generated a novel LAIV candidate strain harboring this mutation. This strain showed higher genetic stability and no ts phenotype reversion. Thus, our high-fidelity strain might be useful for the development of a safer LAIV.

Apical Trafficking Pathways of Influenza A Virus HA and NA via Rab17- and Rab23-Positive Compartments.

The envelope proteins of influenza A virus, hemagglutinin (HA) and neuraminidase (NA), play critical roles in viral entry to host cells and release from the cells, respectively. After protein synthesis, they are transported from the trans-Golgi network (TGN) to the apical plasma membrane (PM) and assembled into virus particles. However, the post-TGN transport pathways of HA and NA have not been clarified. Temporal study by confocal microscopy revealed that HA and NA colocalized soon after their synthesis, and relocated together from the TGN to the upper side of the cell. Using the Rab family protein, we investigated the post-TGN transport pathways of HA and NA. HA partially colocalized with AcGFP-Rab15, Rab17, and Rab23, but rarely with AcGFP-Rab11. When analyzed in cells stably expressing AcGFP-Rab, HA/NA colocalized with Rab15 and Rab17, markers of apical sorting and recycling endosomes, and later colocalized with Rab23, which distributes to the apical PM and endocytic vesicles. Overexpression of the dominant-negative (DN) mutants of Rab15 and Rab17, but not Rab23, significantly delayed HA transport to the PM. However, Rab23DN impaired cell surface expression of HA. Live-cell imaging revealed that NA moved rapidly with Rab17 but not with Rab15. NA also moved with Rab23 in the cytoplasm, but this motion was confined at the upper side of the cell. A fraction of HA was localized to Rab17 and Rab23 double-positive vesicles in the cytoplasm. Coimmunoprecipitation indicated that HA was associated with Rab17 and Rab23 in lipid raft fractions. When cholesterol was depleted by methyl-ß-cyclodextrin treatment, the motion of NA and Rab17 signals ceased. These results suggest that HA and NA are incorporated into lipid raft microdomains and are cotransported to the PM by Rab17-positive and followed by Rab23-positive vesicles.

Local structural changes of the influenza A virus ribonucleoprotein complex by single mutations in the specific residues involved in efficient genome packaging.

The influenza A virus genome consists of eight single-stranded negative-sense RNA segments. The noncoding regions located at the 3'- and 5'- ends of each segment are necessary for genome packaging, and the terminal coding regions are required to precisely bundle the eight segments. However, the nucleotide residues important for genome bundling are not defined. Here, we introduced premature termination codons in the hemagglutinin (HA) or matrix protein 2 (M2) gene and constructed virus libraries containing random sequences in the terminal coding regions. Using these virus libraries, we identified nucleotide residues involved in efficient virus propagation. Viral genome packaging was impaired in viruses that contained single mutations at these identified residues. Furthermore, these single mutations altered the local structure of the viral ribonucleoprotein complex. Our results show that specific nucleotide residues in the viral protein coding region are involved in forming the precise structure of the viral ribonucleoprotein complex.

A C4N4 Diaminopyrimidine Fluorophore.

A new scaffold for producing efficient organic fluorescent materials was identified: 2,5-diamino-4,6-diarylpyrimidine featuring a C4N4 elemental composition. Single-step installation of two aryl groups at the 4,6-positions of the pyrimidine core delivered fluorescent organic materials in a modular fashion. A range of fluorescent compounds with distinct absorption/emission properties was readily accessed by changing the aromatic attachments. A generally high absorption coefficient and quantum yield were observed, including C4N4 derivatives that could fluoresce even in the solid state. The two amino groups at the 2,5-positions of the pyrimidine were essential for intense fluorescence with a large Stokes shift, which was corroborated by structural relaxation to a p-iminoquinone-like structure in the excited state. Besides live-cell imaging capabilities, fluorescent labeling of a protein involved in autophagy elucidated a new protein-protein interaction, supporting potential utility in bioimaging applications.

Flupyranochromene, a novel inhibitor of influenza virus cap-dependent endonuclease, from Penicillium sp. f28743.

Influenza virus RNA polymerase has cap-dependent endonuclease activity that produces capped RNA fragments for priming viral mRNA synthesis. This enzymatic activity is essential for viral propagation, but it is not present in any host cellular enzyme, making it an attractive target for the development of anti-influenza drugs. Here, we isolated a novel inhibitor of cap-dependent endonuclease, named flupyranochromene, from the fermentation broth of the fungus Penicillium sp. f28743. Structural analysis revealed that this compound bears a putative pharmacophore that chelates divalent metal ion(s) present in the endonuclease active site in the PA subunit of the polymerase. Consistently, in vitro endonuclease assays showed that flupyranochromene exerts its inhibitory effects by blocking endonucleolytic cleavage by the PA subunit of viral RNA polymerase complex.

Translation of Hepatitis A Virus IRES Is Upregulated by a Hepatic Cell-Specific Factor.

Many viruses strongly prefer to infect certain cell types, a phenomenon known as "tropism." Understanding tropism's molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5' untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3' end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.

Anti-influenza virus activity of a salcomine derivative mediated by inhibition of viral RNA synthesis.

Influenza virus infection is a major threat to global health. Although vaccines and anti-influenza virus drugs are available, annual influenza virus epidemics result in severe illness, and an influenza pandemic occurs every 20-30 years. To identify candidate anti-influenza virus compounds, we screened approximately 5,000 compounds in an in-house library. We identified MZ7465, a salcomine derivative, as a potent inhibitor of influenza virus propagation. We analyzed the antiviral propagation mechanism of the hit compound by determining the amounts of viral proteins and RNA in infected cells treated with or without the hit compound. Treatment of infected cells with MZ7465 decreased both viral protein and RNA synthesis. In addition, an in vitro assay showed that viral RNA synthesis was directly inhibited by MZ7465. These results suggest that salcomine and its derivatives are potential candidates for the treatment of influenza virus infections.

Current landscape and future prospects of antiviral drugs derived from microbial products.

Viral infections are a major global health threat. Over the last 50 years, significant efforts have been devoted to the development of antiviral drugs and great success has been achieved for some viruses. However, other virus infections, such as epidemic influenza, still spread globally and new threats continue to arise from emerging and re-emerging viruses and drug-resistant viruses. In this review, the contributions of microbial products isolated in Institute of Microbial Chemistry for antiviral research are summarized. In addition, the current state of development of antiviral drugs that target influenza virus and hepatitis B virus, and the future prospects for antivirals from natural products are described and discussed.The Journal of Antibiotics advance online publication, 11 October 2017; doi:10.1038/ja.2017.115.

Encountered-Type Haptic Interface for Representation of Shape and Rigidity of 3D Virtual Objects.

This paper describes the development of an encountered-type haptic interface that can generate the physical characteristics, such as shape and rigidity, of three-dimensional (3D) virtual objects using an array of newly developed non-expandable balloons. To alter the rigidity of each non-expandable balloon, the volume of air in it is controlled through a linear actuator and a pressure sensor based on Hooke's law. Furthermore, to change the volume of each balloon, its exposed surface area is controlled by using another linear actuator with a trumpet-shaped tube. A position control mechanism is constructed to display virtual objects using the balloons. The 3D position of each balloon is controlled using a flexible tube and a string. The performance of the system is tested and the results confirm the effectiveness of the proposed principle and interface.

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases.

Enhancement of Anti-Inflammatory and Osteogenic Abilities of Mesenchymal Stem Cells via Cell-to-Cell Adhesion to Periodontal Ligament-Derived Fibroblasts.

Mesenchymal stem cells (MSCs) are involved in anti-inflammatory events and tissue repair; these functions are activated by their migration or homing to inflammatory tissues in response to various chemokines. However, the mechanism by which MSCs interact with other cell types in inflammatory tissue remains unclear. We investigated the role of periodontal ligament fibroblasts (PDL-Fs) in regulating the anti-inflammatory and osteogenic abilities of bone marrow-derived- (BM-) MSCs. The expression of monocyte chemotactic protein- (MCP-)1 was significantly enhanced by stimulation of PDL-Fs with inflammatory cytokines. MCP-1 induced the migratory ability of BM-MSCs but not PDL-Fs. Expression levels of anti-inflammatory and inflammatory cytokines were increased and decreased, respectively, by direct-contact coculture between MSCs and PDL-Fs. In addition, the direct-contact coculture enhanced the expression of MSC markers that play important roles in the self-renewal and maintenance of multipotency of MSCs, which in turn induced the osteogenic ability of the cells. These results suggest that MCP-1 induces the migration and homing of BM-MSCs into the PDL inflammatory tissue. The subsequent adherence of MSCs to PDL-Fs plays an immunomodulatory role to terminate inflammation during wound healing and upregulates the expression stem cell markers to enhance the stemness of MSCs, thereby facilitating bone formation in damaged PDL tissue.

Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain.

Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft.

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-ß and bone morphogenetic protein signaling.

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG­2, ­3, and ­5 from the bone marrow of green fluorescent protein (GFP)­transgenic mice. These cell lines clearly expressed mouse MSC markers Sca­1 and CD44, and SG­2 and ­5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)­ß superfamily. By contrast, SG­3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG­3 cells, which underwent osteogenesis in response to BMP, while TGF­ß receptor II was most highly expressed in SG­3 and ­5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF­ß1 in SG­2 cells but not in SG­3 or ­5 cells. Furthermore, TGF­ß1 clearly induced the expression of Smad­interacting transcription factor CCAAT/enhancer binding protein­ß in SG­2 but not in SG­3 or ­5 cells. These results demonstrated the establishment of TGF­ß­responsive SG­2 MSCs, BMP­responsive SG­3 MSCs and TGF­ß/BMP­unresponsive SG­5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF­ß superfamily affects the proliferation and differentiation status of MSCs in vivo.