N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA.

Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.

Structure-based discovery of antiviral inhibitors targeting the E dimer interface of Japanese encephalitis virus.

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery.

DDX21 translocates from nucleus to cytoplasm and stimulates the innate immune response due to dengue virus infection.

Successful DENV infection relies on its ability to evade the host innate immune system. By using iTRAQ labeling followed by LC-MS/MS analysis, DDX21 was identified as a new host RNA helicase involved in the DENV life cycle. In DENV infected cells, DDX21 translocates from nucleus to cytoplasm to active the innate immune response and thus inhibits DENV replication in the early stages of infection. DDX21 is then degraded by the viral NS2B-NS3 protease complex and the innate immunity is thus subverted to facilitate DENV replication. The results reveal a new mechanism in which DENV subverts the host innate immune system to facilitate its replication in host cells.

Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1.

Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3' end of dengue virus (DENV) 5'-3' UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

Single dose recombinant VSV based vaccine elicits robust and durable neutralizing antibody against Hantaan virus.

Hantaan virus (HTNV) is a pathogenic orthohantavirus prevalent in East Asia that is known to cause hemorrhagic fever with severe renal syndrome (HFRS), which has a high fatality rate. However, a Food and Drug Administration (FDA)-approved vaccine is not currently available against this virus. Although inactivated vaccines have been certified and used in endemic regions for decades, the neutralizing antibody (NAb) titer induced by inactivated vaccines is low and the immunization schedule is complicated, requiring at least three injections spanning approximately 6 months to 1 year. Replication-competent vesicular stomatitis virus (VSV)-based vaccines provide prolonged protection after a single injection. In this study, we successfully engineered the HTNV glycoprotein (GP) in the VSV genome by replacing the VSV-G open reading frame. The resulting recombinant (r) rVSV-HTNV-GP was rescued, and the immunogenicity of GP was similar to that of HTNV. BALB/c mice immunized with rVSV-HTNV-GP showed a high titer of NAb against HTNV after a single injection. Notably, the cross-reactive NAb response induced by rVSV-HTNV-GP against Seoul virus (an orthohantavirus) was higher than that induced by three sequential injections of inactivated vaccines. Upon challenge with HTNV, rVSV-HTNV-GP-immunized mice showed a profoundly reduced viral burden in multiple tissues, and inflammation in the lungs and liver was nearly undetectable. Moreover, a single injection of rVSV-HTNV-GP established a prolonged immunological memory status as the NAbs were sustained for over 1 year and provided long-term protection against HTNV infection. The findings of our study can support further development of an rVSV-HTNV-GP-based HTNV vaccine with a simplified immunization schedule.

The Comparison of Inflammatory Cytokines (IL-6 and IL-18) and Immune Cells in Japanese Encephalitis Patients With Different Progression.

Background: Japanese encephalitis virus (JEV) is the main cause of viral encephalitis in Asia. Nowadays, no effective and specific therapy for JE patients is available except supportive treatment. The fatality rate of JE patients is still about 30%, and more than half of survivors suffered from various neuropsychiatric sequelae. Thus, more attention should be paid to JE. Methods: In this study, a retrospective cohort of JE patients was collected and the general features of JE patients admitted into the Department of Infectious Diseases were analyzed. Meanwhile, the dynamic change of plasma cytokines and immune cells in JE patients with divergent prognosis was detected and analyzed. Results: We found a mounted proportion of adult/old patients in JE cases. The level of IL-6 and IL-18 increased in JE patients especially in fatal individuals. There was a continuous decreased percentage of CD4+ T and B cells in severe JE patients with fatal outcome compared with the surviving JE patients. Conclusions: The consistent high level of IL-6 and IL-18 in the plasma and low proportion of CD4+ T and B cells in the PBMCs might be the indicators of poor prognosis.

An algal lectin griffithsin inhibits Hantaan virus infection in vitro and in vivo.

Hantaan virus (HTNV) is the etiological pathogen of hemorrhagic fever with renal syndrome in East Asia. There are currently no effective therapeutics approved for HTNV and other hantavirus infections. We found that griffithsin (GRFT), an algae-derived lectin with broad-spectrum antiviral activity against various enveloped viruses, can inhibit the growth and spread of HTNV. In vitro experiments using recombinant vesicular stomatitis virus (rVSV) with HTNV glycoproteins as a model revealed that the GRFT inhibited the entry of rVSV-HTNV-G into host cells. In addition, we demonstrated that GRFT prevented authentic HTNV infection in vitro by binding to the viral N-glycans. In vivo experiments showed that GRFT partially protected the suckling mice from death induced by intracranial exposure to HTNV. These results demonstrated that GRFT can be a promising agent for inhibiting HTNV infection.

miR-155 regulates pro- and anti-inflammatory cytokine expression in human monocytes during chronic hepatitis C virus infection.

BACKGROUND: Hepatitis C virus (HCV) dysregulates innate and adaptive immune responses while monocytes (M) play a crucial role in linking innate and adaptive immunity to control viral infection. A transcription factor T-bet is upregulated to dampen M functions via the c-Jun N-terminal kinase (JNK) pathway, followed by enhanced Tim-3 expression in chronic HCV infection. However, the molecular mechanisms that control the expression in M are yet unknown. miR-155 has been implicated as a key regulator controlling diverse biological processes through posttranscriptional repression, but the influences of miR-155 on these regulators and effectors still need to be studied. METHODS: Forty HCV-infected patients and 40 healthy subjects (HS) were recruited, THP-1 cells (human acute monocyte leukemia cell line) were cultured with HCV-infected Huh 7.5 cells. The expression levels of miR-155 and JNK1/JNK2/JNK3 were measured by real-time RT-PCR. IL-10/IL-12 was detected by flow cytometry. THP-1 cells were transfected with mimics-155 and negative control, SOCS1, p-STAT1, p65, p-smad, p-p38, and p-JNK were measured by Western blot. TNF-α levels were measured by ELISA. Student's t-test was used in statistics. RESULTS: The study showed that miR-155 was upregulated in CD14+ M in HCV-infected patients compared to healthy subjects (P<0.05). Moreover, the upregulation of miR-155 in CD14+ M from HCV-infected patients induced TNF-α production and JNK gene expression, which, in turn, led to T-bet upregulation. Also, miR-155 upregulation in CD14+ M of HCV-infected patients increased the IL-12 and decreased the IL-10 production. CONCLUSIONS: The obtained results indicated that miR-155 upregulation in M during HCV infection enhances the activation of TNF-α and JNK pathways, promotes the expression of transcription factor T-bet, and triggers pro- and anti-inflammatory mediators. Together, these data reveal new information regarding the mechanisms of chronic HCV infection.

Integrated Metabolomics and Transcriptomics Analyses Reveal Metabolic Landscape in Neuronal Cells during JEV Infection.

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis in endemic regions of Asia. The neurotropism of JEV and its high-efficiency replication in neurons are the key events for pathogenesis. Revealing the interplay between virus and host cells in metabolic facet is of great importance both for unraveling the pathogenesis mechanisms and providing novel antiviral targets. This study took advantage of the integration analysis of metabolomics and transcriptomics to depict the metabolic profiles of neurons during the early stage of JEV infection. Increased glycolysis and its branched pentose phosphate pathway (PPP) flux and impaired oxidative phosphorylation (OXPHOS) in glucose utilization, and the catabolic patterns of lipid metabolism were created to facilitate the biosynthesis of precursors needed for JEV replication in neurons. Pharmacological inhibitions of both glycolysis pathway and PPP in neurons suggested its indispensable role in maintaining the optimal propagation of JEV. In addition, analysis of metabolomic-transcriptomic regulatory network showed the pivotal biological function of lipid metabolism during JEV infection. Several pro-inflammatory lipid metabolites were significantly up-regulated and might partially be responsible for the progression of encephalitis. These unique metabolic reprogramming features might give deeper insight into JEV infected neurons and provide promising antiviral approaches targeting metabolism.

Corrigendum: An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection.

[This corrects the article DOI: 10.3389/fphar.2019.01203.].

RIPK3 Promotes JEV Replication in Neurons via Downregulation of IFI44L.

Japanese encephalitis virus (JEV), the leading cause of viral encephalitis in Asia, is neurovirulent and neuroinvasive. Neurons are the main target of JEV infection and propagation. Receptor interacting serine/threonine-protein kinase 3 (RIPK3) has been reported to contribute to neuroinflammation and neuronal death in many central nervous system diseases. In this study, we found that the progression of JE was alleviated in RIPK3-knockout (RIPK3-/-) mice in both peripheral and intracerebral infection. RIPK3-knockdown (RIPK3-RNAi) neuro2a cells showed higher cell viability during JEV infection. Moreover, the JEV load was significantly decreased in RIPK3-/- mouse-derived primary neurons and RIPK3-RNAi neuro2a cells compared with wild-type neurons, but this was not observed in microglia. Furthermore, RNA sequencing of brain tissues showed that the level of the interferon (IFN)-induced protein 44-like gene (IFI44L) was significantly increased in JEV-infected RIPK3-/- mouse brains, RIPK3-/- neurons, and RIPK3-RNAi-neuro2a cells. Then, it was demonstrated that the propagation of JEV was inhibited in IFI44L-overexpressing neuro2a cells and enhanced in IFI44L and RIPK3 double knockdown neuro2a cells. Taken together, our results showed that the increased expression of RIPK3 following JEV infection played complicated roles. On the one hand, RIPK3 participated in neuroinflammation and neuronal death during JEV infection. On the other hand, RIPK3 inhibited the expression of IFI44L to some extent, leading to the propagation of JEV in neurons, which might be a strategy for JEV to evade the cellular innate immune response.

Corrigendum: Remdesivir (GS-5734) Impedes Enterovirus Replication Through Viral RNA Synthesis Inhibition.

[This corrects the article DOI: 10.3389/fmicb.2020.01105.].

Remdesivir (GS-5734) Impedes Enterovirus Replication Through Viral RNA Synthesis Inhibition.

Human enteroviruses are responsible for diverse diseases, from mild respiratory symptoms to fatal neurological complications. Currently, no registered antivirals have been approved for clinical therapy. Thus, a therapeutic agent for the enterovirus-related disease is urgently needed. Remdesivir (GS-5734) is a novel monophosphoramidate adenosine analog prodrug that exhibits potent antiviral activity against diverse RNA virus families, including positive-sense Coronaviridae and Flaviviridae and negative-sense Filoviridae, Paramyxoviridae, and Pneumoviridae. Currently, remdesivir is under phase 3 clinical development for disease COVID-19 treatment. Here, we found that remdesivir impeded both EV71 viral RNA (vRNA) and complementary (cRNA) synthesis, indicating that EV71 replication is inhibited by the triphosphate (TP) form of remdesivir. Moreover, remdesivir showed potent antiviral activity against diverse enteroviruses. These data extend the remdesivir antiviral activity to enteroviruses and indicate that remdesivir is a promising antiviral treatment for EV71 and other enterovirus infections.

An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection.

Hantaviruses, etiologic pathogens responsible for two severe human diseases, exist in areas ranging from Eurasia to America and remain global public health concerns. Conventionally, plaque formation assays have been used for hantavirus titering. However, hantaviruses replicate slowly within cells and produce minimal cytopathic effects, making this technique difficult to master. The improved enzyme-linked immunosorbent assay-based antigen detection method is easier to perform but is still time consuming. Here, we established an enzyme-linked focus formation assay (FFA) for Hantaan virus titering that is twice as fast as traditional assays. Moreover, using this method, we evaluated the effects of favipiravir (T-705) and another influenza virus drug, baloxavir acid (BXA), on hantavirus replication. We found that the endonuclease inhibitor BXA exerted similar anti-hantavirus effects as T-705. Overall, we developed a time-saving method for hantavirus titering and suggest BXA as a potential treatment choice for hantavirus-exposed individuals.

Mesenchymal stem cells alleviate Japanese encephalitis virus-induced neuroinflammation and mortality.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. Japanese encephalitis (JE) caused by JEV is characterized by extensive inflammatory cytokine secretion, microglia activation, blood-brain barrier (BBB) breakdown, and neuronal death, all of which contribute to the vicious cycle of inflammatory damage. There are currently no effective treatments for JE. Mesenchymal stem cells (MSCs) have been demonstrated to have a therapeutic effect on many central nervous system (CNS) diseases by regulating inflammation and other mechanisms. METHODS: In vivo, 8- to 10-week-old mice were infected intraperitoneally with JEV and syngeneic bone marrow MSCs were administered through the caudal vein at 1 and 3 days post-infection. The mortality, body weight, and behavior were monitored daily. Brains from each group were harvested at the indicated times for hematoxylin and eosin staining, immunohistochemical observation, flow cytometric analysis, TUNEL staining, Western blot, quantitative real-time polymerase chain reaction, and BBB permeability assays. In vitro, co-culture and mixed culture experiments of MSCs with either microglia or neurons were performed, and then the activation state of microglia and survival rate of neurons were tested 48 h post-infection. RESULTS: MSC treatment reduced JEV-induced mortality and improved the recovery from JE in our mouse model. The inflammatory response, microglia activation, neuronal damage, BBB destruction, and viral load (VL) were significantly decreased in the MSC-treated group. In co-culture experiments, MSCs reprogrammed M1-to-M2 switching in microglia and improved neuron survival. Additionally, the VL was decreased in Neuro2a cells in the presence of MSCs accompanied by increased expression of interferon-α/ß. CONCLUSION: MSC treatment alleviated JEV-induced inflammation and mortality in mice.

MLKL Mediated Necroptosis Accelerates JEV-Induced Neuroinflammation in Mice.

Japanese encephalitis virus (JEV) is the most prevalent cause of viral encephalitis in Asia and the western Pacific. Neuronal death caused by JEV infection and inflammation induced cytotoxicity leads to progression and deterioration of Japanese encephalitis (JE). Mixed-lineage kinase domain-like protein (MLKL) mediated necroptosis is a newly discovered pathway of programmed cell death and participates in many inflammatory diseases. In this study, we demonstrated for the first time that necroptosis was involved in the neuronal loss during JE via immune-electron microscopy and immunochemistry. The expression of MLKL in neurons was upregulated in presence of JEV infection in vitro and in vivo. Deletion of MLKL alleviated the progression of JE and decreased the level of inflammatory cytokines in mice model. Taken together, this study provides evidence for the participation of necroptosis in the pathogenesis of JEV infection.

Interferon-α-Enhanced CD100/Plexin-B1/B2 Interactions Promote Natural Killer Cell Functions in Patients with Chronic Hepatitis C Virus Infection.

BACKGROUND: CD100, also known as Sema4D, is an immune semaphorin constitutively expressed on natural killer (NK) cells and T cells. As an immune activation molecule, CD100 has important immunoregulatory effects on NK functions by enhancing the interactions between NK cells and target cells. The aim of this study was to investigate whether hepatitis C virus (HCV) infection affects CD100 expression, and whether interferon-α treatment enhances NK killing activity to facilitate HCV clearance via CD100. METHODS: Expression of CD100 on NK cells was evaluated by flow cytometry in patients with chronic HCV infection, with or without pegylated interferon-α-based therapy. NK cell cytotoxicity and interferon (IFN)-γ production were measured by flow cytometry upon culturing the NK cells with K562 and Huh7.5 or HCV JFH-1-infected Huh7.5 cells. RESULTS: The frequency of CD100+ NK cells in HCV-infected individuals was slightly suppressed compared to healthy subjects. IFN-α treatment could significantly upregulate CD100 expression, which was confirmed by in vitro studies using peripheral blood mononuclear cells cocultured with HCV-expressing Huh7.5 cells or IFN-α. Importantly, the expression of CD100 on NK cells from HCV patients was inversely associated with the HCV-RNA levels in the early phase of IFN-α therapy, and the IFN-α upregulated CD100 led to an enhanced NK killing activity through ligations with its receptors plexin-B1/B2 on target cells. CONCLUSION: These results implied a novel mechanism by which IFN-α enhanced CD100/Plexin-B1/B2 interaction plays an important role in promoting NK functions in patients with chronic hepatitis C.

[Alterations and clinical signifecance of exosome-containing innate immunity related lncRNAs in patients of hemorrhagic fever with renal syndrome].

Objective To observe the alterations of innate immunity related long non-coding RNAs (lncRNAs) in exosomes extracted from the plasma of hemorrhagic fever with renal syndrome (HFRS) patients, and analyze their relationship with the disease stage and severity. Methods Exosomes were extracted from the plasma samples of HFRS patients, healthy controls and recovered HFRS patients. Transmission electronic microscopy and Western blotting were performed to confirm the efficiency of the extraction. lncRNA profiles in the different groups were determined by high-throughput sequencing. The contents of several innate immunity related lncRNAs were detected by quantitative real-time PCR, and their relationship with the disease stage and severity was analyzed. Results Exosomes from the plasma were accurately extracted. Innate immunity related lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), negative regulator of interferon response (NRIR), negative regulator of antiviral response (NRAV) were found in exosomes. NEAT1 content was significantly reduced in the exosomes from HFRS patients compared with healthy controls and it was significantly restored in recovered HFRS patients. The exosome NEAT1 content was correlated with the epidemic of HFRS but had no relationship with the stage and severity of the disease. Conclusion Several innate immunity related lncRNAs exist in the exosome from HFRS patients, among which NEAT1 content significantly decreases in HFRS patients compared with healthy controls and recovered HFRS patients. The reduced NEAT1 level is correlated with the epidemic of HFRS.