Viral RNA capping: Mechanisms and antiviral therapy.

RNA capping is an essential trigger for protein translation in eukaryotic cells. Many viruses have evolved various strategies for initiating the translation of viral genes and generating progeny virions in infected cells via synthesizing cap structure or stealing the RNA cap from nascent host messenger ribonucleotide acid (mRNA). In addition to protein translation, a new understanding of the role of the RNA cap in antiviral innate immunity has advanced the field of mRNA synthesis in vitro and therapeutic applications. Recent studies on these viral RNA capping systems have revealed startlingly diverse ways and molecular machinery. A comprehensive understanding of how viruses accomplish the RNA capping in infected cells is pivotal for designing effective broad-spectrum antiviral therapies. Here we systematically review the contemporary insights into the RNA-capping mechanisms employed by viruses causing human and animal infectious diseases, while also highlighting its impact on host antiviral innate immune response. The therapeutic applications of targeting RNA capping against viral infections and the development of RNA-capping inhibitors are also summarized.

Transcriptome analysis of two tobacco varieties with contrast resistance to Meloidogyne incognita in response to PVY MSNR infection.

Root-knot nematode (RKN) disease is a major disease of tobacco worldwide, which seriously hinders the improvement of tobacco yield and quality. Obvious veinal necrosis-hypersensitive responses are observed only in RKN-resistant lines infected by Potyvirus Y (PVY) MSNR, making this an effective approach to screen for RKN-resistant tobacco. RNA-seq analysis, real-time quantitative PCR (qRT-PCR) and functional enrichment analysis were conducted to gain insight into the transcription dynamics difference between G28 (RKN-resistant) and CBH (RKN-susceptible) varieties infected with PVY MSNR. Results showed that a total of 7900, 10576, 9921, 11530 and 12531 differentially expressed genes (DEGs) were identified between the two varieties at 0, 1, 3, 5, and 7 d after infection, respectively. DEGs were associated with plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis, and photosynthesis-related metabolic pathways. Additional DEGs related to starch and sucrose metabolism, energy production, and the indole-3-acetic acid signaling pathway were induced in CBH plants after infection. DEGs related to phenylpropanoid biosynthesis, abscisic acid, salicylic acid, brassinosteroids, and jasmonic acid signaling pathway were induced in G28 after infection. Our findings reveal DEGs that may contribute to differences in PVY MSNR resistance among tobacco varieties. These results help us to understand the differences in transcriptional dynamics and metabolic processes between RKN-resistant and RKN-susceptible varieties involved in tobacco-PVY MSNR interaction.

Structure-Based Design and Characterization of the Highly Potent and Selective Covalent Inhibitors Targeting the Lysine Methyltransferases G9a/GLP.

Protein lysine methyltransferases G9a and GLP, which catalyze mono- and di-methylation of histone H3K9 and nonhistone proteins, play important roles in diverse cellular processes. Overexpression or dysregulation of G9a and GLP has been identified in various types of cancer. Here, we report the discovery of a highly potent and selective covalent inhibitor 27 of G9a/GLP via the structure-based drug design approach following structure-activity relationship exploration and cellular potency optimization. Mass spectrometry assays and washout experiments confirmed its covalent inhibition mechanism. Compound 27 displayed improved potency in inhibiting the proliferation and colony formation of PANC-1 and MDA-MB-231 cell lines and exhibited enhanced potency in reducing the levels of H3K9me2 in cells compared to noncovalent inhibitor 26. In vivo, 27 showed significant antitumor efficacy in the PANC-1 xenograft model with good safety. These results clearly indicate that 27 is a highly potent and selective covalent inhibitor of G9a/GLP.

Genomic analyses provide insights into the genome evolution and environmental adaptation of the tobacco moth Ephestia elutella.

Ephestia elutella is a major pest responsible for significant damage to stored tobacco over many years. Here, we conduct a comparative genomic analysis on this pest, aiming to explore the genetic bases of environmental adaptation of this species. We find gene families associated with nutrient metabolism, detoxification, antioxidant defense and gustatory receptors are expanded in the E. elutella genome. Detailed phylogenetic analysis of P450 genes further reveals obvious duplications in the CYP3 clan in E. elutella compared to the closely related species, the Indianmeal moth Plodia interpunctella. We also identify 229 rapidly evolving genes and 207 positively selected genes in E. elutella, respectively, and highlight two positively selected heat shock protein 40 (Hsp40) genes. In addition, we find a number of species-specific genes related to diverse biological processes, such as mitochondria biology and development. These findings advance our understanding of the mechanisms underlying processes of environmental adaptation on E. elutella and will enable the development of novel pest management strategies.

Electrochemical Healing of Fractured Metals.

Repairing fractured metals to extend their useful lifetimes advances sustainability and mitigates carbon emissions from metal mining and processing. While high-temperature techniques are being used to repair metals, the increasing ubiquity of digital manufacturing and "unweldable" alloys, as well as the integration of metals with polymers and electronics, call for radically different repair approaches. Herein, a framework for effective room-temperature repair of fractured metals using an area-selective nickel electrodeposition process refered to as electrochemical healing is presented. Based on a model that links geometric, mechanical, and electrochemical parameters to the recovery of tensile strength, this framework enables 100% recovery of tensile strength in nickel, low-carbon steel, two "unweldable" aluminum alloys, and a 3D-printed difficult-to-weld shellular structure using a single common electrolyte. Through a distinct energy-dissipation mechanism, this framework also enables up to 136% recovery of toughness in an aluminum alloy. To facilitate practical adoption, this work reveals scaling laws for the energetic, financial, and time costs of healing, and demonstrates the restoration of a functional level of strength in a fractured standard steel wrench. Empowered with this framework, room-temperature electrochemical healing can open exciting possibilities for the effective, scalable repair of metals in diverse applications.

Cloning and functional analysis of the root-knot nematode resistance gene NtRk1 in tobacco.

Tobacco (Nicotiana tabacum L.) is an economically important crop worldwide. Root-knot nematodes (RKNs) are responsible for yield losses in tobacco and other crops, such as tomato, potato, peanut, and soybean. Therefore, screening for resistance genes that can prevent RKN infestation and the associated damage is crucial. However, there is no report of cloning tobacco RKN resistance genes to date. Here, we cloned the tobacco RKN resistance gene NtRk1 from the resistant variety TI706, using rapid amplification of cDNA ends. NtRk1 has high homology with other RKN resistance genes (CaMi in pepper, Mi-1.1 and Mi-1.2 in tomato). Under normal conditions, NtRk1 was barely expressed in the roots; however, following RKN infection, its expression level rapidly increased. Overexpression of NtRk1 in the susceptible cultivar "Changbohuang" enhanced its resistance to Meloidogyne incognita, while RNA interference of NtRk1 in the resistant cultivar K326 resulted in its susceptibility to M. incognita. Moreover, compared with resistant variety K326, we found the salicylic acid and jasmonic acid contents of RNAi plants decreased after inoculation with M. incognita, and confirmed that the function of NtRk1 is related to these phytohormones. These findings indicate that NtRk1 is an RKN resistance gene, which is abundantly expressed in response to RKN infection and may enhance host defense responses by elevating salicylic acid and jasmonic acid levels.

Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability.

Intestinal stem cells (ISCs) at the crypt base are responsible for the regeneration of the intestinal epithelium. However, how ISC self-renewal is regulated still remains unclear. Here we identified a circular RNA, circBtnl1, that is highly expressed in ISCs. Loss of circBtnl1 in mice enhanced ISC self-renewal capacity and epithelial regeneration, without changes in mRNA and protein levels of its parental gene Btnl1. Mechanistically, circBtnl1 and Atf4 mRNA competitively bound the ATP-dependent RNA helicase Ddx3y to impair the stability of Atf4 mRNA in wild-type ISCs. Furthermore, ATF4 activated Sox9 transcription by binding to its promoter via a unique motif, to enhance the self-renewal capacity and epithelial regeneration of ISCs. In contrast, circBtnl1 knockout promoted Atf4 mRNA stability and enhanced ATF4 expression, which caused Sox9 transcription to potentiate ISC stemness. These data indicate that circBtnl1-mediated Atf4 mRNA decay suppresses Sox9 transcription that negatively modulates self-renewal maintenance of ISCs.

Characterization of the complete chloroplast genome of Veronica arvensis and its phylogenomic inference in plantaginaceae.

Veronica arvensis, which is an annual flowering plant in the plantain family Plantaginaceae, has commonly used as a Chinese herbal medicine to treat malaria in China. Here, the complete plastome of V. arvensis was successfully assembled based on genome skimming sequencing. The plastome of V. arvensis was 149,386 bp in length, comprising a pair of inverted repeats (IR; 24,946 bp) separated by a large single-copy (LSC) region (82,004 bp) and a small single-copy (SSC) region (17,490 bp). The plastid genome encoded 113 unique genes, consisting of 79 protein-coding genes, 30 tRNA genes, and four rRNA genes, with 19 duplicated genes in the IR regions. Six plastid hotspot regions (trnH-psbA, trnK-rps16, atpI-rps2, ndhF-rpl32, ccsA-ndhD and rps15-ycf1) were identified within Veronica. Phylogenetic analysis showed that the representative species from Veronica was monophyletic. V. persica and V. polita formed a maximum clade, followed by sister to V. arvensis.

SRSF5-Mediated Alternative Splicing of M Gene is Essential for Influenza A Virus Replication: A Host-Directed Target Against Influenza Virus.

Splicing of influenza A virus (IAV) RNA is an essential process in the viral life cycle that involves the co-opting of host factors. Here, it is demonstrated that induction of host serine and arginine-rich splicing factor 5 (SRSF5) by IAV facilitated viral replication by enhancing viral M mRNA splicing. Mechanistically, SRSF5 with its RRM2 domain directly bounds M mRNA at conserved sites (M mRNA position 163, 709, and 712), and interacts with U1 small nuclear ribonucleoprotein (snRNP) to promote M mRNA splicing and M2 production. Mutations introduced to the three binding sites, without changing amino acid code, significantly attenuates virus replication and pathogenesis in vivo. Likewise, SRSF5 conditional knockout in the lung protects mice against lethal IAV challenge. Furthermore, anidulafungin, an approved antifungal drug, is identified as an inhibitor of SRSF5 that effectively blocks IAV replication in vitro and in vivo. In conclusion, SRSF5 as an activator of M mRNA splicing promotes IAV replication and is a host-derived antiviral target.

Influenza virus NS1 interacts with 14-3-3ε to antagonize the production of RIG-I-mediated type I interferons.

For influenza A viruses (IAVs), non-structural protein 1 (NS1) protein was recognized to be the key factor to enhance virulence by antagonizing host innate anti-viral responses. However, for the pathways allowing NS1 to regulate the type I interferon (IFN) response, the identification of the substrates was still incomplete. Here a recombinant IAV encoding a NS1 containing an affinity tag (NS1-Strep) was generated to capture the NS1-interactome in the lungs of infected mice. Several scaffold proteins of the 14-3-3 family were distinguished as the most potent candidates. Based on the conserved motif RxxTxxT of NS1, the interaction between NS1 and 14-3-3ε was enabled, which competed for the binding of RIG-I to 14-3-3ε and prevented RIG-I translocation to the adaptor MAVS, consequently inhibiting IFN-ß expression. A recombinant mutant IAV deficient in 14-3-3ε binding elicited a markable innate immune responses and showed impaired growth kinetics.

p21 restricts influenza A virus by perturbing the viral polymerase complex and upregulating type I interferon signaling.

Many cellular genes and networks induced in human lung epithelial cells infected with the influenza virus remain uncharacterized. Here, we find that p21 levels are elevated in response to influenza A virus (IAV) infection, which is independent of p53. Silencing, pharmacological inhibition or deletion of p21 promotes virus replication in vitro and in vivo, indicating that p21 is an influenza restriction factor. Mechanistically, p21 binds to the C-terminus of IAV polymerase subunit PA and competes with PB1 to limit IAV polymerase activity. Besides, p21 promotes IRF3 activation by blocking K48-linked ubiquitination degradation of HO-1 to enhance type I interferons expression. Furthermore, a synthetic p21 peptide (amino acids 36 to 43) significantly inhibits IAV replication in vitro and in vivo. Collectively, our findings reveal that p21 restricts IAV by perturbing the viral polymerase complex and activating the host innate immune response, which may aid the design of desperately needed new antiviral therapeutics.

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans.

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

Characterization and in vitro assessment of three-dimensional extrusion Mg-Sr codoped SiO2-complexed porous microhydroxyapatite whisker scaffolds for biomedical engineering.

BACKGROUND: Large bone defects have always been a great challenge for orthopedic surgeons. The use of a good bone substitute obtained by bone tissue engineering (BTE) may be an effective treatment method. Artificial hydroxyapatite, a commonly used bone defect filler, is the main inorganic component of bones. Because of its high brittleness, fragility, and lack of osteogenic active elements, its application is limited. Therefore, its fragility should be reduced, its osteogenic activity should be improved, and a more suitable scaffold should be constructed. METHODS: In this study, a microhydroxyapatite whisker (mHAw) was developed, which was doped with the essential trace active elements Mg2+ and Sr2+ through a low-temperature sintering technique. After being formulated into a slurry, a bionic porous scaffold was manufactured by extrusion molding and freeze drying, and then SiO2 was used to improve the mechanical properties of the scaffold. The hydrophilicity, pore size, surface morphology, surface roughness, mechanical properties, and release rate of the osteogenic elements of the prepared scaffold were detected and analyzed. In in vitro experiments, Sprague-Dawley (SD) rat bone marrow mesenchymal stem cells (rBMSCs) were cultured on the scaffold to evaluate cytotoxicity, cell proliferation, spreading, and osteogenic differentiation. RESULTS: Four types of scaffolds were obtained: mHAw-SiO2 (SHA), Mg-doped mHAw-SiO2 (SMHA), Sr-doped mHAw-SiO2 (SSHA), and Mg-Sr codoped mHAw-SiO2 (SMSHA). SHA was the most hydrophilic (WCA 5°), while SMHA was the least (WCA 8°); SMHA had the smallest pore size (247.40 ± 23.66 µm), while SSHA had the largest (286.20 ± 19.04 µm); SHA had the smallest Young's modulus (122.43 ± 28.79 MPa), while SSHA had the largest (188.44 ± 47.89 MPa); and SHA had the smallest compressive strength (1.72 ± 0.29 MPa), while SMHA had the largest (2.47 ± 0.25 MPa). The osteogenic active elements Si, Mg, and Sr were evenly distributed and could be sustainably released from the scaffolds. None of the scaffolds had cytotoxicity. SMSHA had the highest supporting cell proliferation and spreading rate, and its ability to promote osteogenic differentiation of rBMSCs was also the strongest. CONCLUSIONS: These composite porous scaffolds not only have acceptable physical and chemical properties suitable for BTE but also have higher osteogenic bioactivity and can possibly serve as potential bone repair materials.

A Nearly Packaging-Free Design Paradigm for Light, Powerful, and Energy-Dense Primary Microbatteries.

Billions of internet connected devices used for medicine, wearables, and robotics require microbattery power sources, but the conflicting scaling laws between electronics and energy storage have led to inadequate power sources that severely limit the performance of these physically small devices. Reported here is a new design paradigm for primary microbatteries that drastically improves energy and power density by eliminating the vast majority of the packaging and through the use of high-energy-density anode and cathode materials. These light (50-80 mg) and small (20-40 µL) microbatteries are enabled though the electroplating of 130 µm-thick 94% dense additive-free and crystallographically oriented LiCoO2 onto thin metal foils, which also act as the encapsulation layer. These devices have 430 Wh kg-1 and 1050 Wh L-1 energy densities, 4 times the energy density of previous similarly sized microbatteries, opening up the potential to power otherwise unpowerable microdevices.

Centimetre-scale crack-free self-assembly for ultra-high tensile strength metallic nanolattices.

Nanolattices exhibit attractive mechanical, energy conversion and optical properties, but it is challenging to fabricate large nanolattices while maintaining the dense regular nanometre features that enable their properties. Here we report a crack-free self-assembly approach for fabricating centimetre-scale nickel nanolattices with much larger crack-free areas than prior self-assembled nanolattices and many more unit cells than three-dimensionally printed nanolattices. These nickel nanolattices have a feature size of 100 nm, a grain size of 30 nm and a tensile strength of 260 MPa, which approaches the theoretical strength limit for porous nickel. The self-assembly method and porous metal mechanics reported in this work may advance the fabrication and applications of high-strength multifunctional porous materials.

IFI16 directly senses viral RNA and enhances RIG-I transcription and activation to restrict influenza virus infection.

The retinoic acid-inducible gene I (RIG-I) receptor senses cytoplasmic viral RNA and activates type I interferons (IFN-I) and downstream antiviral immune responses. How RIG-I binds to viral RNA and how its activation is regulated remains unclear. Here, using IFI16 knockout cells and p204-deficient mice, we demonstrate that the DNA sensor IFI16 enhances IFN-I production to inhibit influenza A virus (IAV) replication. IFI16 positively upregulates RIG-I transcription through direct binding to and recruitment of RNA polymerase II to the RIG-I promoter. IFI16 also binds to influenza viral RNA via its HINa domain and to RIG-I protein with its PYRIN domain, thus promoting IAV-induced K63-linked polyubiquitination and RIG-I activation. Our work demonstrates that IFI16 is a positive regulator of RIG-I signalling during influenza virus infection, highlighting its role in the RIG-I-like-receptor-mediated innate immune response to IAV and other RNA viruses, and suggesting its possible exploitation to modulate the antiviral response.

Reassortment with dominant chicken H9N2 influenza virus contributed to the fifth H7N9 virus human epidemic.

H9N2 Avian influenza virus (AIV) is regarded as a principal donor of viral genes through reassortment to co-circulating influenza viruses that can result in zoonotic reassortants. Whether H9N2 virus can maintain sustained evolutionary impact on such reassortants is unclear. Since 2013, avian H7N9 virus had caused five sequential human epidemics in China; the fifth wave in 2016-2017 was by far the largest but the mechanistic explanation behind the scale of infection is not clear. Here, we found that, just prior to the fifth H7N9 virus epidemic, H9N2 viruses had phylogenetically mutated into new sub-clades, changed antigenicity and increased its prevalence in chickens vaccinated with existing H9N2 vaccines. In turn, the new H9N2 virus sub-clades of PB2 and PA genes, housing mammalian adaptive mutations, were reassorted into co-circulating H7N9 virus to create a novel dominant H7N9 virus genotype that was responsible for the fifth H7N9 virus epidemic. H9N2-derived PB2 and PA genes in H7N9 virus conferred enhanced polymerase activity in human cells at 33°C and 37°C, and increased viral replication in the upper and lower respiratory tracts of infected mice which could account for the sharp increase in human cases of H7N9 virus infection in the 2016-2017 epidemic. The role of H9N2 virus in the continual mutation of H7N9 virus highlights the public health significance of H9N2 virus in the generation of variant reassortants of increasing zoonotic potential.IMPORTANCEAvian H9N2 influenza virus, although primarily restricted to chicken populations, is a major threat to human public health by acting as a donor of variant viral genes through reassortment to co-circulating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role, as donor of new sub-clade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (G72) with enhanced infectivity in humans during the 2016-2017 N7N9 virus epidemic. Our findings emphasize that the ongoing evolution of prevalent H9N2 virus in chickens is an important source, via reassortment, of mammalian adaptive genes for other influenza virus subtypes. Thus, close monitoring of prevalence and variants of H9N2 virus in chicken flocks is necessary in the detection of zoonotic mutations.

The matrix gene of pdm/09 H1N1 contributes to the pathogenicity and transmissibility of SIV in mammals.

The H1N1 influenza virus of swine-origin was responsible for the H1N1 pandemic in 2009 (pdm/09 H1N1), where the virus was transmitted to humans and then spread between people, and its continued circulation has resulted in it becoming a seasonal human flu virus. Since 2016, the matrix (M) gene of pdm/09 H1N1 has been involved in the reassortment of swine influenza viruses (SIVs) in China and has gradually become a dominant genotype in pigs. However, whether M gene substitution will influence the fitness of emerging SIVs remains unclear. Here, we analyzed the biological characteristics of SIVs with the M gene from Eurasian avian-like (EA) SIV or pdm/09 H1N1 in mammals and found that SIVs containing the pdm/09-M gene exhibit stronger virulence in mice, more efficient respiratory droplet transmission between ferrets, and increased transcription of viral genes in A549 cells compared with those containing EA-M. We also determined the functional significance of the pdm/09-M gene in conferring an elevated release of progeny viruses comprised of largely filamentous virions rather than spherical virions. Our study suggests that pdm/09-M plays a crucial role in the genesis of emerging SIVs in terms of the potential prevalence in the population.

Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus.

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG's antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.