Effect of moringa extract on parasitemia, monocyte activation and organomegaly among Mus musculus infected by Plasmodium berghei ANKA.

In Indonesia, malaria remains a problem, with 94,610 active cases in 2021 and its current therapy includes chloroquine and artemisinin; however, resistance has been commonly reported. To overcome this problem, studies about potential medicinal plants that can be used as antimalaria, such as moringa (Moringa oleifera) started to receive more attention. The aim of this study was to investigate the effects of moringa in parasitemia, monocyte activation, and organomegaly on animal model malaria. This experimental study used male Mus musculus, infected by Plasmodium berghei ANKA, as an animal malaria model. The extract was made by maceration of dry moringa leaves, which were then divided into three concentrations: 25%, 50%, and 75%. Dihydroartemisinin-piperazine was used as a positive control treatment, and distilled water as a negative control treatment. The animals were observed for six days to assess the parasitemia count and the number of monocyte activation. On day 7, the animals were terminated, and the liver, spleen, and kidney were weighed. The results showed that the effective concentrations in reducing parasitemia and inducing monocyte activation were 50% and 25% of moringa leaf extract, respectively. The smallest liver and spleen enlargement was observed among animals within the group treated with a 50% concentration of M. oleifera extract. In contrast, the smallest kidney enlargement was observed in the group treated with 25% of M. oleifera extract. Further analysis is recommended to isolate compounds with antimalarial properties in moringa leaves.

Novel Anti-Viral Properties of the Herbal Extract of Davallia mariesii against Influenza A Virus.

Gu-Sui-Bu, the dried rhizome of Davallia mariesii, is a traditional Chinese herbal remedy with a significant history of treating osteoporosis and inflammatory conditions. However, its potential as an anti-influenza agent and its underlying mechanisms of action remain unexplored. To obtain a more potent extract from D. mariesii and gain insights into its mechanism of action against influenza A virus (IAV), we utilized a partitioning process involving organic solvents and water, resulting in the isolation of butanolic subfractions of the D. mariesii extract (DMBE). DMBE exhibited a broad anti-viral spectrum, effectively inhibiting IAV, with an EC50 of 24.32 ± 6.19 µg/mL and a selectivity index of 6.05. We subsequently conducted a series of in vitro assays to evaluate the antiviral effects of DMBE and to uncover its mechanisms of action. DMBE was found to inhibit IAV during the early stages of infection by hindering the attachment of the virus onto and its penetration into host cells. Importantly, DMBE was observed to hinder IAV-mediated cell-cell fusion. It also inhibited neuraminidase activity, plaque size, and the expression levels of phospho-AKT. In summary, this study provides evidence for the effectiveness of D. mariesii as a complementary and alternative herbal remedy against IAV. Specifically, our data highlight DMBE's capabilities in inhibiting viral entry and the release of virions.

BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Natural compounds inhibit Monkeypox virus methyltransferase VP39 in silico studies.

VP39, an essential 2'-O-RNA methyltransferase enzyme discovered in Monkeypox virus (MPXV), plays a vital role in viral RNA replication and transcription. Inhibition of the enzyme may prevent viral replication. In this context, using a combination of molecular docking and molecular dynamics (MDs) simulations, the inhibitory ability of NCI Diversity Set VII natural compounds to VP39 protein was investigated. It should be noted that the computed binding free energy of ligand via molecular docking and linear interaction energy (LIE) approaches are in good agreement with the corresponding experiments with coefficients of R=0.72 and 0.75, respectively. NSC 319990, NSC 196515 and NSC 376254 compounds were demonstrated that can inhibit MPVX methyltransferase VP39 protein with the similar affinity compared to available inhibitor sinefungin. Moreover, nine residues involving Gln39, Gly68, Gly72, Asp95, Arg97, Val116, Asp138, Arg140 and Asn156 may be argued that they play an important role in binding process of inhibitors to VP39.Communicated by Ramaswamy H. Sarma.

Anti-viral and Anti-inflammatory Isoflavonoids from Ukrainian Iris aphylla Rhizomes: Structure-Activity Relationship Coupled with ChemGPS-NP Analysis.

Dried Iris rhizomes have been used in Chinese and European traditional medicine for the treatment of various diseases such as bacterial infections, cancer, and inflammation, as well as for being astringent, laxative, and diuretic agents. Eighteen phenolic compounds including some rare secondary metabolites, such as irisolidone, kikkalidone, irigenin, irisolone, germanaism B, kaempferol, and xanthone mangiferin, were isolated for the first time from Iris aphylla rhizomes. The hydroethanolic Iris aphylla extract and some of its isolated constituents showed protective effects against influenza H1N1 and enterovirus D68 and anti-inflammatory activity in human neutrophils. The promising anti-influenza effect of apigenin (13: , almost 100% inhibition at 50 µM), kaempferol (14: , 92%), and quercetin (15: , 48%) were further confirmed by neuraminidase inhibitory assay. Irisolidone (1: , almost 100% inhibition at 50 µM), kikkalidone (5: , 93%), and kaempferol (14: , 83%) showed promising anti-enterovirus D68 activity in vitro. The identified compounds were plotted using ChemGPS-NP to correlate the observed activity of the isolated phenolic compounds with the in-house database of anti-influenza and anti-enterovirus agents. Our results indicated that the hydroethanolic Iris aphylla extract and Iris phenolics hold the potential to be developed for the management of seasonal pandemics of influenza and enterovirus infections.

SARS-CoV-2 pandemics: An update of CRISPR in diagnosis and host-virus interaction studies.

Since December 2019, the Coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread rapidly around the world, overburdening healthcare systems and creating significant global health concerns. Rapid detection of infected individuals via early diagnostic tests and administration of effective therapy remains vital in pandemic control, and recent advances in the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system may support the development of novel diagnostic and therapeutic approaches. Cas-based SARS-CoV-2 detection methods (FnCAS9 Editor Linked Uniform Detection Assay (FELUDA), DNA endonuclease-targeted CRISPR trans reporter (DETECTR), and Specific High-sensitivity Enzymatic Reporter Unlocking (SHERLOCK)) have been developed for easier handling compared to quantitative polymerase chain reaction (qPCR) assays, with good rapidity, high specificity, and reduced need for complex instrumentation. Cas-CRISPR-derived RNA (Cas-crRNA) complexes have been shown to reduce viral loads in the lungs of infected hamsters, by degrading virus genomes and limiting viral replication in host cells. Viral-host interaction screening platforms have been developed using the CRISPR-based system to identify essential cellular factors involved in pathogenesis, and CRISPR knockout (CRISPRKO) and activation screening results have revealed vital pathways in the life cycle of coronaviruses, including host cell entry receptors (ACE2, DPP4, and ANPEP), proteases involved in spike activation and membrane fusion (cathepsin L (CTSL) and transmembrane protease serine 2 (TMPRSS2)), intracellular traffic control routes for virus uncoating and budding, and membrane recruitment for viral replication. Several novel genes (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin, subfamily A, member 4 (SMARCA4), ARIDIA, and KDM6A) have also been identified via systematic data mining analysis as pathogenic factors for severe CoV infection. This review highlights how CRISPR-based systems can be applied to investigate the viral life cycle, detect viral genomes, and develop therapies against SARS-CoV-2 infection.

Rosmarinic acid interferes with influenza virus A entry and replication by decreasing GSK3ß and phosphorylated AKT expression levels.

BACKGROUND: The purpose of this study was to examine the in vivo activity of rosmarinic acid (RA) - a phytochemical with antioxidant, anti-inflammatory, and antiviral properties - against influenza virus (IAV). An antibody-based kinase array and different in vitro functional assays were also applied to identify the mechanistic underpinnings by which RA may exert its anti-IAV activity. METHODS: We initially examined the potential efficacy of RA using an in vivo mouse model. A time-of-addition assay and an antibody-based kinase array were subsequently applied to investigate mechanism-of-action targets for RA. The hemagglutination inhibition assay, neuraminidase inhibition assay, and cellular entry assay were also performed. RESULTS: RA increased survival and prevented body weight loss in IAV-infected mice. In vitro experiments revealed that RA inhibited different IAV viruses - including oseltamivir-resistant strains. From a mechanistic point of view, RA downregulated the GSK3ß and Akt signaling pathways - which are known to facilitate IAV entry and replication into host cells. CONCLUSIONS: RA has promising preclinical efficacy against IAV, primarily by interfering with the GSK3ß and Akt signaling pathways.

3,4-Dicaffeoylquinic Acid from the Medicinal Plant Ilex kaushue Disrupts the Interaction Between the Five-Fold Axis of Enterovirus A-71 and the Heparan Sulfate Receptor.

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.

Identification of Potential Drug Targets of Broad-Spectrum Inhibitors with a Michael Acceptor Moiety Using Shotgun Proteomics.

The Michael addition reaction is a spontaneous and quick chemical reaction that is widely applied in various fields. This reaction is performed by conjugating an addition of nucleophiles with α, ß-unsaturated carbonyl compounds, resulting in the bond formation of C-N, C-S, C-O, and so on. In the development of molecular materials, the Michael addition is not only used to synthesize chemical compounds but is also involved in the mechanism of drug action. Several covalent drugs that bond via Michael addition are regarded as anticarcinogens and anti-inflammatory drugs. Although drug development is mainly focused on pharmaceutical drug discovery, target-based discovery can provide a different perspective for drug usage. However, considerable time and labor are required to define a molecular target through molecular biological experiments. In this review, we systematically examine the chemical structures of current FDA-approved antiviral drugs for potential Michael addition moieties with α, ß-unsaturated carbonyl groups, which may exert an unidentified broad-spectrum inhibitory mechanism to target viral or host factors. We thus propose that profiling the targets of antiviral agents, such as Michael addition products, can be achieved by employing a high-throughput LC-MS approach to comprehensively analyze the interaction between drugs and targets, and the subsequent drug responses in the cellular environment to facilitate drug repurposing and/or identify potential adverse effects, with a particular emphasis on the pros and cons of this shotgun proteomic approach.

Anti-Inflammatory, Antiallergic, and COVID-19 Main Protease (Mpro) Inhibitory Activities of Butenolides from a Marine-Derived Fungus Aspergillus terreus.

In December 2020, the U.K. authorities reported to the World Health Organization (WHO) that a new COVID-19 variant, considered to be a variant under investigation from December 2020 (VUI-202012/01), was identified through viral genomic sequencing. Although several other mutants were previously reported, VUI-202012/01 proved to be about 70% more transmissible. Hence, the usefulness and effectiveness of the newly U.S. Food and Drug Administration (FDA)-approved COVID-19 vaccines against these new variants are doubtfully questioned. As a result of these unexpected mutants from COVID-19 and due to lack of time, much research interest is directed toward assessing secondary metabolites as potential candidates for developing lead pharmaceuticals. In this study, a marine-derived fungus Aspergillus terreus was investigated, affording two butenolide derivatives, butyrolactones I (1) and III (2), a meroterpenoid, terretonin (3), and 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde (4). Chemical structures were unambiguously determined based on mass spectrometry and extensive 1D/2D NMR analyses experiments. Compounds (1-4) were assessed for their in vitro anti-inflammatory, antiallergic, and in silico COVID-19 main protease (Mpro) and elastase inhibitory activities. Among the tested compounds, only 1 revealed significant activities comparable to or even more potent than respective standard drugs, which makes butyrolactone I (1) a potential lead entity for developing a new remedy to treat and/or control the currently devastating and deadly effects of COVID-19 pandemic and elastase-related inflammatory complications.

Perilla (Perilla frutescens) leaf extract inhibits SARS-CoV-2 via direct virus inactivation.

BACKGROUND: While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents with mild or no symptoms in most cases, a significant number of patients become critically ill. Remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19) in several countries, but its use as monotherapy has not substantially lowered mortality rates. Because agents from traditional Chinese medicine (TCM) have been successfully utilized to treat pandemic and endemic diseases, we designed the current study to identify novel anti-SARS-CoV-2 agents from TCM. METHODS: We initially used an antivirus-induced cell death assay to screen a panel of herbal extracts. The inhibition of the viral infection step was investigated through a time-of-drug-addition assay, whereas a plaque reduction assay was carried out to validate the antiviral activity. Direct interaction of the candidate TCM compound with viral particles was assessed using a viral inactivation assay. Finally, the potential synergistic efficacy of remdesivir and the TCM compound was examined with a combination assay. RESULTS: The herbal medicine Perilla leaf extract (PLE, approval number 022427 issued by the Ministry of Health and Welfare, Taiwan) had EC50 of 0.12 ± 0.06 mg/mL against SARS-CoV-2 in Vero E6 cells - with a selectivity index of 40.65. Non-cytotoxic PLE concentrations were capable of blocking viral RNA and protein synthesis. In addition, they significantly decreased virus-induced cytokine release and viral protein/RNA levels in the human lung epithelial cell line Calu-3. PLE inhibited viral replication by inactivating the virion and showed additive-to-synergistic efficacy against SARS-CoV-2 when used in combination with remdesivir. CONCLUSION: Our results demonstrate for the first time that PLE is capable of inhibiting SARS-CoV-2 replication by inactivating the virion. Our data may prompt additional investigation on the clinical usefulness of PLE for preventing or treating COVID-19.

Regulation of the proteostasis network during enterovirus infection: A feedforward mechanism for EV-A71 and EV-D68.

The proteostasis network guarantees successful protein synthesis, folding, transportation, and degradation. Mounting evidence has revealed that this network maintains proteome integrity and is linked to cellular physiology, pathology, and virus infection. Human enterovirus A71 (EV-A71) and EV-D68 are suspected causative agents of acute flaccid myelitis, a severe poliomyelitis-like neurologic syndrome with no known cure. In this context, further clarification of the molecular mechanisms underlying EV-A71 and EV-D68 infection is paramount. Here, we summarize the components of the proteostasis network that are intercepted by EV-A71 and EV-D68, as well as antivirals that target this network and may help develop improved antiviral drugs.

Rosmarinic acid exhibits broad anti-enterovirus A71 activity by inhibiting the interaction between the five-fold axis of capsid VP1 and cognate sulfated receptors.

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Design, Synthesis, and Biological Evaluation of Itaconic Acid Derivatives as Potential Anti-Influenza Agents.

Influenza A viruses (IAVs) have caused worldwide epidemics and pandemics by reassortment and generation of drug-resistant mutants, which render antivirals and current vaccinations no longer usable. In this study, an itaconic acid derivative 1 was identified from a chemical library of 20 000 compounds, by performing a cell-based screening assay, as a lead agent exhibiting anti-influenza A activity. Accordingly, a series of itaconic acid derivatives were designed and synthesized by adopting a rational design strategy to obtain more potent anti-influenza agents. The results of an in vitro pharmacological study showed that compounds 4 and 8 exhibited the most potent anti-IAV effect with half-maximal effective concentration values of 0.14 and 0.11 µM, respectively, in Madin-Darby canine kidney cells. The mechanism of action studies showed that lead agents 1 and 4 reduced virus replication by directly targeting IAV nucleoproteins and disrupting virus ribonucleoprotein export from the nucleus to the cytosol. On the basis of its high potential as an anti-IAV agent and its selectivity index >785, compound 4 was found to be a promising candidate for further development against IAVs.

Large-Scale Proteomic Identification of Targets of Cellular miR-197 Downregulated by Enterovirus A71.

MicroRNAs are noncoding RNA species comprising 18-23 nucleotides that regulate host-virus interaction networks. Here, we show that enterovirus A71 infection in human rhabdomyosarcoma (RD) is regulated by miR-197 expression. Transfection of miR-197 mimic into RD cells inhibited virus replication by interfering with the viral RNA synthesis. We employed a combination of mass-spectrometry-based quantitative proteomics with the stable isotope labeling with amino acids in cell culture (SILAC) approach for the identification of the miR-197 target genes in RD cells and to investigate the differential expression of the prospective target proteins. A total of 1822 proteins were repeatedly identified in miR-197-transfected RD cells, 106 of which were predicted to have seed sites by TargetScan. Notably, seven of eight selected genes potentially related to viral replication and immune response were validated as direct miR-197 targets, using a luciferase 3'-untranslated region (UTR) reporter assay. The expression levels of three selected endogenous molecules (ITGAV, ETF1, and MAP2K1/MEK1) were significantly reduced when RD cells were transfected with a miR-197 mimic. Our results provide a comprehensive database of miR-197 targets, which might provide better insights into the understanding of host-virus interaction.

Design, synthesis & structure-activity relationships of a new class of antihuman enterovirus D68 & A71 agents.

AIM: No antiviral medications are currently approved to treat enterovirus (EV)-associated disease or prevent EV infection. METHODS: In this study, a series of probenecid derivatives were designed via a rational strategy and synthesized to obtain more potent anti-EV agents. RESULTS: Compounds 8 and 24 exhibited the most potent activity against EV D68 and A71, with half maximal effective concentration (EC50) values of 2.49/2.09 and 2.59/2.41 µM, respectively, and revealed a broad inhibition spectrum toward other EV strains, with high selectivity indices. Additionally, compounds 8 and 24 showed good stability in rat serum, with half-lives of 48.39 and 60.26 min, respectively. CONCLUSION: Compounds 8 and 24 are the promising candidates for the development of new agents against EV D68 and A71 viruses.

A pilot study on primary cultures of human respiratory tract epithelial cells to predict patients' responses to H7N9 infection.

Avian influenza A(H7N9) virus infections frequently lead to acute respiratory distress syndrome and death in humans. We aimed to investigate whether primary cultures of human respiratory tract epithelial cells are helpful to understand H7N9 virus pathogenesis and tissue tropism, and to evaluate how patient-related characteristics can affect the host's response to infection. Normal human bronchial epithelial cells (isolated from two different donors) and primary epithelial cells (harvested from 27 patients undergoing airway surgery) were experimentally infected with H7N9 and/or H1N1pdm for 72 h. After virus infection, the culture media were collected for viral RNA quantitation and cytokine detection. Both H7N9 and H1N1pdm viruses replicated and induced a cytokine response differently for each donor in the normal human bronchial epithelial model. H7N9 replicated equivalently in epithelial cells harvested from the inferior turbinate and paranasal sinus, and those from the larynx and bronchus, at 72 h post-infection. Viral RNA quantity at 72 h was significantly higher in patients aged 21-64 years than in patients aged ≥ 65 years; however, no effects of sex, medical comorbidities, and obesity were noted. H7N9-infected cultured cells released multiple cytokines within 72 h. Levels of interleukin-1ß, interleukin-6, interleukin-8, interferon-γ, and tumor necrosis factor-α were associated differently with patient-related characteristics (such as age, sex, obesity, and medical comorbidities). In the era of precision medicine, these findings illustrate the potential utility of this primary culture approach to predict a host's response to H7N9 infection or to future infection by newly emerging viral infections, and to dissect viral pathogenesis.

A novel role of ER stress signal transducer ATF6 in regulating enterovirus A71 viral protein stability.

BACKGROUND: Due to limited coding capacity of viral genome, enterovirus A71 (EV-A71) co-opts host nuclear proteins for its replication. Upon ER stress, the ER-localized 90 kDa activating transcription factor 6 (p90ATF6) is proteolytically cleaved to produce the transcriptionally active amino-terminal 50 kDa (p50ATF6) product where it enters the nucleus to activate a subset of unfolded protein response and ER-associated degradation (also known as ERAD) genes. During EV-A71 infection, however, this p50ATF6 product was not detected in the nucleus, and its downstream target genes were not activated. METHODS: We examined the role of ATF6 during EV-A71 infection, including its cleavage process and its role in viral life cycle by silencing or overexpressing ATF6. RESULTS: We showed that a potential cleavage in the middle of p90ATF6 produced an amino-terminal ~ 45 kDa fragment in a viral protease-independent but EV-A71-dependent manner. The disappearance of ATF6 was not restricted to a specific strain of EV-A71 or cell type, and was not simply caused by picornavirus-mediated global translational shutoff. This cleavage of ATF6, which was most likely mediated by the host response, was nevertheless independent of both cellular caspases and XBP1-associated proteasomes. The silencing of ATF6 expression by small interfering RNA suppressed viral titers due to reduced viral protein stability. This effect was markedly restored by the ectopic expression of p90ATF6. CONCLUSION: Our findings indicate that ATF6 plays a distinct role in viral protein stability and that the host uses different cleavage strategies, rather than conventional cleavage by generating p50ATF6, to combat viral infection.

dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication.

Enterovirus 71 (EV-A71) causes hand, foot and mouth disease in young children and infants, but can also cause severe neurological complications or even death. The double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), an interferon-induced antiviral protein, phosphorylates the regulatory α-subunit of the eukaryotic translation initiation factor 2 in response to viral infection, thereby blocking the translation of cellular and viral mRNA and promoting apoptosis. The cleavage of PKR after infection with poliovirus, a prototype enterovirus, has been reported by others, but the underlying mechanism of this cleavage and its role in viral replication remain unclear. In the present study, we show that viral 3C protease cleaves PKR at a site, Q188, which differs from the site cleaved during apoptosis, D251. In contrast to the conventional phosphorylation of PKR by dsRNA, EV-A71 3C physically interacts with PKR to mediate the phosphorylation of PKR; this effect is dependent on 3C protease activity. Overexpression of a catalytically inactive PKR mutant (K296H) accelerates viral protein accumulation and increases virus titer, whereas a K64E substitution in the dsRNA binding site abolishes this advantage. We also demonstrate that PKR cleavage mediated by EV-A71 3C protease produces a short N-terminal PKR fragment that can enhance EV-A71 replication, in terms of viral RNA, viral protein, and viral titers. We conclude that PKR is co-opted by EV-A71 via viral protease 3C-mediated proteolytic activation to facilitate viral replication.