Structure of the Ebola virus polymerase complex.

Filoviruses, including Ebola virus, pose an increasing threat to the public health. Although two therapeutic monoclonal antibodies have been approved to treat the Ebola virus disease1,2, there are no approved broadly reactive drugs to control diverse filovirus infection. Filovirus has a large polymerase (L) protein and the cofactor viral protein 35 (VP35), which constitute the basic functional unit responsible for virus genome RNA synthesis3. Owing to its conservation, the L-VP35 polymerase complex is a promising target for broadly reactive antiviral drugs. Here we determined the structure of Ebola virus L protein in complex with tetrameric VP35 using cryo-electron microscopy (state 1). Structural analysis revealed that Ebola virus L possesses a filovirus-specific insertion element that is essential for RNA synthesis, and that VP35 interacts extensively with the N-terminal region of L by three protomers of the VP35 tetramer. Notably, we captured the complex structure in a second conformation with the unambiguous priming loop and supporting helix away from polymerase active site (state 2). Moreover, we demonstrated that the century-old drug suramin could inhibit the activity of the Ebola virus polymerase in an enzymatic assay. The structure of the L-VP35-suramin complex reveals that suramin can bind at the highly conserved NTP entry channel to prevent substrates from entering the active site. These findings reveal the mechanism of Ebola virus replication and may guide the development of more powerful anti-filovirus drugs.

Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement.

Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions.

Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity.

Induction and suppression of antiviral RNA interference (RNAi) has been observed in mammals during infection with at least seven distinct RNA viruses, including some that are pathogenic in humans. However, while the cell-autonomous immune response mediated by antiviral RNAi is gradually being recognized, little is known about systemic antiviral RNAi in mammals. Furthermore, extracellular vesicles (EVs) also function in viral signal spreading and host immunity. Here, we show that upon antiviral RNAi activation, virus-derived small-interfering RNAs (vsiRNAs) from Nodamura virus (NoV), Sindbis virus (SINV), and Zika virus (ZIKV) enter the murine bloodstream via EVs for systemic circulation. vsiRNAs in the EVs are biologically active, since they confer RNA-RNA homology-dependent antiviral activity in both cultured cells and infant mice. Moreover, we demonstrate that vaccination with a live-attenuated virus, rendered deficient in RNAi suppression, induces production of stably maintained vsiRNAs and confers protective immunity against virus infection in mice. This suggests that vaccination with live-attenuated VSR (viral suppressor of RNAi)-deficient mutant viruses could be a new strategy to induce immunity.

Actomyosin-dependent cell contractility orchestrates Zika virus infection.

Emerging pathogen infections, such as Zika virus (ZIKV), pose an increasing threat to human health, but the role of mechanobiological attributes of host cells during ZIKV infection is largely unknown. Here, we reveal that ZIKV infection leads to increased contractility of host cells. Importantly, we investigated whether host cell contractility contributes to ZIKV infection efficacy, from both the intracellular and extracellular perspective. By performing drug perturbation and gene editing experiments, we confirmed that disruption of contractile actomyosin compromises ZIKV infection efficiency, viral genome replication and viral particle production. By culturing on compliant matrix, we further demonstrate that a softer substrate, leading to less contractility of host cells, compromises ZIKV infection, which resembles the effects of disrupting intracellular actomyosin organization. Together, our work provides evidence to support a positive correlation between host cell contractility and ZIKV infection efficacy, thus unveiling an unprecedented layer of interplay between ZIKV and the host cell.

KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

UBC9-mediated SUMOylation of Lamin B1 enhances DNA-damage-induced nuclear DNA leakage and autophagy after spinal cord injury.

Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI). Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence. In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death. Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.

Fast Nano-IR Hyperspectral Imaging Empowered by Large-Dataset-Free Miniaturized Spatial-Spectral Network.

The emerging field of nanoscale infrared (nano-IR) offers label-free molecular contrast, yet its imaging speed is limited by point-by-point traverse acquisition of a three-dimensional (3D) data cube. Here, we develop a spatial-spectral network (SS-Net), a miniaturized deep-learning model, together with compressive sampling to accelerate the nano-IR imaging. The compressive sampling is performed in both the spatial and spectral domains to accelerate the imaging process. The SS-Net is trained to learn the mapping from small nano-IR image patches to the corresponding spectra. With this elaborated mapping strategy, the training can be finished quickly within several minutes using the subsampled data, eliminating the need for a large-labeled dataset of common deep learning methods. We also designed an efficient loss function, which incorporates the image and spectral similarity to enhance the training. We first validate the SS-Net on an open stimulated Raman-scattering dataset; the results exhibit the potential of 10-fold imaging speed improvement with state-of-the-art performance. We then demonstrate the versatility of this approach on atomic force microscopy infrared (AFM-IR) microscopy with 7-fold imaging speed improvement, even on nanoscale Fourier transform infrared (nano-FTIR) microscopy with up to 261.6 folds faster imaging speed. We further showcase the generalization of this method on AFM-force volume-based multiparametric nanoimaging. This method establishes a paradigm for rapid nano-IR imaging, opening new possibilities for cutting-edge research in materials, photonics, and beyond.

Neferine alleviates acute kidney injury by regulating the PPAR-α/NF-κB pathway.

Acute kidney injury (AKI) is a cluster of clinical syndromes with diverse etiologies that ultimately result in a swift decline in kidney function. Regrettably, AKI lacks effective therapeutic agents at present. Neferine, a bioactive alkaloid derived from Lotus Plumule, has been reported to alleviate AKI triggered by cisplatin, ischemia/reperfusion (I/R), and sepsis by inhibiting inflammatory pathways. However, the precise molecular mechanisms underpinning its renoprotective effects remain elusive. Peroxisome proliferator-activated receptor alpha (PPAR-α), a regulator of lipid metabolism with anti-inflammatory properties, was investigated in this study to examine its role in neferine's renoprotective effects in cellular and mouse models of AKI. We found that neferine pretreatment in both I/R- or lipopolysaccharide (LPS)-induced AKI models inhibited the activation of the NF-κB inflammatory pathway and reversed PPAR-α deficiency. In NRK-52E cells exposed to hypoxia/reoxygenation (H/R) or LPS, overexpression of PPAR-α resulted in inhibition of the NF-κB pathway and TNF-α production, while PPAR-α silencing via siRNA transfection negated neferine's anti-inflammatory effects. Furthermore, pretreatment with neferine not only reduced lipid accumulation but also reversed the downregulation of FAO-related enzymes induced by LPS. Our findings suggest that neferine's renoprotective effects against AKI are partially mediated through the reversal of renal PPAR-α deficiency and subsequent inhibition of the inflammatory NF-κB pathway. Therefore, regulating renal PPAR-α expression by neferine could represent a promising therapeutic strategy for AKI.

Environmental Impact Assessment for the Use of an Orally Aerosolized Adenovirus Type-5 Vector-Based COVID-19 Vaccine in Randomized Clinical Trials.

BACKGROUND: An orally aerosolized adenovirus type-5 vector-based coronavirus disease 2019 (COVID-19) vaccine (Ad5-nCoV) has recently been authorized for boosting immunization in China. Our study aims to assess the environmental impact of the use of aerosolized Ad5-nCoV. METHODS: We collected air samples from rooms, swabs from the desks on which the vaccine nebulizer was set, mask samples from participants, and blood samples of nurses who administered the inoculation in the clinical trials. The viral load of adenovirus type-5 vector in the samples and the antibody levels against the wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain in serum were detected. RESULTS: Only one (4.00%) air sample collected before initiation of vaccination was positive and most air samples collected during and after vaccination were positive (97.96%, 100%, respectively). All nurses in trial A showed at least 4-fold increase of the neutralizing antibody against SARS-CoV-2 after initiation of the study. In trial B, the proportion of positive mask samples was 72.97% at 30 minutes after vaccination, 8.11% at day 1, and 0% at days 3, 5, and 7. CONCLUSIONS: Vaccination with the orally aerosolized Ad5-nCoV could result in some spillage of the vaccine vector viral particles in the environment and cause human exposure. Clinical Trials Registration. NCT04840992 and NCT05303584.

Efficient Production of 3-Amino-2-Hydroxy Acetophenone by Multi-Enzyme Biosynthesis.

We developed a synthetic route for producing 3-amino-2-hydroxy acetophenone (3AHAP) from m-nitroacetophenone (3NAP) using an in vitro approach. Various reaction systems were evaluated, and a direct reaction method with crude enzyme and supersaturated substrates for optimal catalytic efficiency was chosen. The reaction system included three enzymes and was enhanced by adjusting enzyme molar ratios and optimizing ribosomal binding sites. We performed substrate docking and alanine scanning to identify key sites in the enzymes nitrobenzene nitroreductase (nbzA) and hydroxylaminobenzene mutase (habA). The optimal mutant was obtained through site-directed mutagenesis, and incorporated into the reaction system, resulting in increased product yield. After optimization, the yield of 3AHAP increased from 75 mg/L to 580 mg/L within 5 hours, the highest reported yield using biosynthesis. This work provides a promising strategy for the efficient and sustainable production of 3AHAP, which has critical applications in the chemical and pharmaceutical industries.

KLF14/miR-1283/TFAP2C axis inhibits HER2-positive breast cancer progression via declining tumor cell proliferation.

MiR-1283 has been identified as a tumor suppressor in some malignancies. Whereas, the role of miR-1283 in HER2-positive (HER2+) breast cancer, particularly its role in regulating cell proliferation, one of the most significant features of tumor progression, is unclear. The related microRNA screened by the breast cancer sample GSE131599 dataset were detected in HER2+ breast cancer tissues and cell lines. Then, the obtained miR-1283 was overexpressed in SKBR3 and BT-474 cells followed by relevant functional assays concerning cell proliferation and apoptosis. The xenograft mouse model was induced and the effect of miR-1283 on tumor growth and cell proliferation was examined. The target of miR-1283 and the transcription factor regulating miR-1283 were predicted and identified. Finally, the influence of transcription factor KLF14 on cell proliferation and apoptosis was investigated. An integrated analysis confirmed that miR-1283 expression was significantly decreased in HER2+ breast cancer tissues. Also, by q-RT-PCR detection, miR-1283 expression was markedly reduced in HER2+ breast cancer tissues and cell lines. The miR-1283 overexpression prevented the proliferation and enhanced apoptosis of HER2+ breast cancer cells, as well as inhibited tumor growth. Mechanistically, miR-1283 inhibited TFAP2C expression by targeting the 3'-untranslated regions of TFAP2C messenger RNA, and the KLF14 enhanced miR-1283 level via binding to its promoter. The result subsequently confirmed the KLF14/miR-1283 signaling suppressed cell proliferation in HER2+ breast cancer. Our results suggested that the KLF14/miR-1283/TFAP2C axis inhibited HER2+ breast cancer progression, which might provide novel insight into mechanical exploration for this disease.

Glycometabolism regulates hepatitis C virus release.

HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response.

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption.

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

A novel cell culture system modeling the SARS-CoV-2 life cycle.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry.

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.

The First Lanthipeptide from Lactobacillus iners, Inecin L, Exerts High Antimicrobial Activity against Human Vaginal Pathogens.

Vaginal infections continue to be a serious public health issue, and developing new approaches to address antibiotic-resistant pathogens is an urgent task. The dominant vaginal Lactobacillus species and their active metabolites (e.g., bacteriocins) have the potential to defeat pathogens and help individuals recover from disorders. Here, we describe for the first time a novel lanthipeptide, inecin L, a bacteriocin from Lactobacillus iners with posttranslational modifications. The biosynthetic genes of inecin L were actively transcribed in the vaginal environment. Inecin L was active against the prevailing vaginal pathogens, such as Gardnerella vaginalis and Streptococcus agalactiae, at nanomolar concentrations. We demonstrated that the antibacterial activity of inecin L was closely related to the N terminus and the positively charged His13 residue. In addition, inecin L was a bactericidal lanthipeptide that showed little effect on the cytoplasmic membrane but inhibited the cell wall biosynthesis. Thus, the present work characterizes a new antimicrobial lanthipeptide from a predominant species of the human vaginal microbiota. IMPORTANCE The human vaginal microbiota plays essential roles in preventing pathogenic bacteria, fungi, and viruses from invading. The dominant vaginal Lactobacillus species show great potential to be developed as probiotics. However, the molecular mechanisms (such as bioactive molecules and their modes of action) involved in the probiotic properties remain to be determined. Our work describes the first lanthipeptide molecule from the dominant Lactobacillus iners. Additionally, inecin L is the only lanthipeptide found among the vaginal lactobacilli thus far. Inecin L shows strong antimicrobial activity toward the prevalent vaginal pathogens and antibiotic-resistant strains, suggesting that inecin L is a potent antibacterial molecule for drug development. In addition, our results show that inecin L exhibits specific antibacterial activity related to the residues in the N-terminal region and ring A, which will contribute to structure-activity relationship studies in lacticin 481-like lanthipeptides.

Food and gut originated bacteriocins involved in gut microbe-host interactions.

The gut microbes interact with each other as well as host, influencing human health and some diseases. Many gut commensals and food originated bacteria produce bacteriocins which can inhibit pathogens and modulate gut microbiota. Bacteriocins have comparable narrow antimicrobial spectrum and are attractive potentials for precision therapy of gut disorders. In this review, the bacteriocins from food and gut microbiomes and their involvement in the interaction between producers and gut ecosystem, along with their characteristics, types, biosynthesis, and functions are described and discussed. Bacteriocins are produced by many intestinal commensals and food microbes among which lactic acid bacteria (many are probiotics) has been paid more attention. Bacteriocin production has been generally regarded as a probiotic trait. They give a competitive advantage to bacteria, enabling their colonization in human gut, and mediating the interaction between the producers and host ecosystem. They fight against unwanted bacteria and pathogens without significant impact on the composition of commensal microbiota. Bacteriocins assist the producers to survive and colonize in the gut microbial populations. There is a great need to evaluate and utilize the potential of bacteriocins for improved therapeutic implications for intestinal health.

Identification and characterization of Sofosbuvir-resistant mutations of hepatitis C virus genotype 3a replicon.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Among its 8 genotypes (GT), GT3 has a relatively lower sustained virological response to highly effective direct-acting antiviral agents (DAA). Sofosbuvir (SOF), an anti-NS5B polymerase inhibitor, is a core component of many anti-HCV DAA cocktail regimens, and its resistant mutations are rare in clinics because these mutations usually severely impair the NS5B polymerase activity, including a mutation S282T in NS5B, the most frequently reported SOF-resistant mutation. In this study, we selected SOF-resistant variants of a previously developed GT3 subgenomic replicon (PR87A7). Two mutations were identified in the viral genome of SOF-resistant PR87A7 variants, Q606R in nontargeted NS3 and S282T in targeted N5SB. We demonstrated that Q606R could rescue the replication defect of S282T in PR87A7, and the resulting double mutant confers the SOF resistance. Finally, we showed that NS3-606R could not compensate for the replication defect of S282T in other GTs. In conclusion, we identified a novel GT3-specific combination of two mutations that confers SOF resistance. Our result calls for attention to potential mutations that may arise in nontargeted viral proteins during the SOF-based DAA treatment of chronic HCV.

Construction and characterization of a new hepatitis C virus genotype 6a subgenomic replicon that is prone to render the sofosbuvir resistance.

Hepatitis C virus (HCV) infection remains a challenge to human public health despite the development of highly effective direct-acting antivirals (DAAs). Sofosbuvir (SOF), a key component in most DAA-based anti-HCV cocktail regimens, is a potent viral RNA polymerase (NS5B) inhibitor with a high barrier to drug resistance. The serine-to-threonine mutation at NS5B 282 (S282T) confers the SOF resistance, but severely impairs viral replication in most HCV genotypes (GTs) and cannot be stably maintained after the termination of the SOF-based therapies. In this study, we first developed a new HCV GT-6a subgenomic replicon PR58D6. Next, we selected SOF-resistant PR58D6 variants by culturing the replicon cells in the presence of SOF. Interestingly, unlike many other HCV replicons which require additional mutations to compensate for the S282T-inducing fitness loss, S282T alone in PR58D6 is genetically stable and confers the SOF resistance without significantly impairing viral replication. Furthermore, we showed that amino acid residue at NS5B 74 (R74) and 556 (D556) which are conserved in GT 6a HCV contribute to efficient replication of PR58D6 containing S282T. Finally, we showed that the G556D mutation in NS5B could rescue the replication deficiency of the S282T in JFH1, a GT-2a replicon. In conclusion, we showed that a novel GT-6a HCV replicon may easily render SOF resistance, which may call for attention to potential drug resistance during DAA therapies of HCV GT-6a patients.

Inhibiting the transcription and replication of Ebola viruses by disrupting the nucleoprotein and VP30 protein interaction with small molecules.

Ebola virus (EBOV) causes hemorrhagic fever in humans with high morbidity and fatality. Although over 45 years have passed since the first EBOV outbreak, small molecule drugs are not yet available. Ebola viral protein VP30 is a unique RNA synthesis cofactor, and the VP30/NP interaction plays a critical role in initiating the transcription and propagation of EBOV. Here, we designed a high-throughput screening technique based on a competitive binding assay to bind VP30 between an NP-derived peptide and a chemical compound. By screening a library of 8004 compounds, we obtained two lead compounds, Embelin and Kobe2602. The binding of these compounds to the VP30-NP interface was validated by dose-dependent competitive binding assay, surface plasmon resonance, and thermal shift assay. Moreover, the compounds were confirmed to inhibit the transcription and replication of the Ebola genome by a minigenome assay. Similar results were obtained for their two respective analogs (8-gingerol and Kobe0065). Interestingly, these two structurally different molecules exhibit synergistic binding to the VP30/NP interface. The antiviral efficacy (EC50) increased from 1 µM by Kobe0065 alone to 351 nM when Kobe0065 and Embelin were combined in a 4:1 ratio. The synergistic anti-EBOV effect provides a strong incentive for further developing these lead compounds in future studies.