Anti-hepatitis B virus activity of lithospermic acid, a polyphenol from Salvia miltiorrhiza, in vitro and in vivo by autophagy regulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza (the roots of S. miltiorrhiza Bunge, Danshen in Chinese), a traditional Chinese medicine, has been clinically used to prevent and treat various diseases, such as cardiovascular and cerebrovascular diseases, diabetes, and hepatitis B, in China and some other Asian countries. Lithospermic acid (LA), a polyphenol derived from S. miltiorrhiza, has been reported to exhibit multiple pharmacological properties, such as anti-inflammatory, anti-HIV, and anti-carbon tetrachloride-induced liver injury activities. However, little is known about the anti-hepatitis B virus (HBV) activity of LA. AIM OF THE STUDY: The study was projected to investigate the anti-HBV activity of LA in vitro (HepG2.2.15 and pHBV1.3-transfected HepG2 cells) and in vivo (pAAV-HBV1.2 hydrodynamic injection [HBV-HDI] mice) and explore the potential mechanism as well. MATERIALS AND METHODS: Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) contents were detected by ELISA kits. HBV DNA and hepatitis B core antigen (HBcAg) levels were evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry assay, respectively. The proteins in autophagy process, lysosomal acidic function, and autophagy-related signaling pathways were examined by Western blot. Transmission electron microscopy was used to observe the number of autophagosomes and autolysosomes. Confocal microscopy was applied to analyze the autophagic flux and lysosomal acidification, using mCherry-enhanced green fluorescent protein (EGFP)-microtubule-associated protein light chain (LC)3 and lysosomal probes, respectively. RESULTS: LA exhibited anti-HBV activity by inhibiting HBV DNA replication in HepG2.2.15 and pHBV-transfected HepG2 cells in dose- and time-dependent manners and hampering HBsAg and HBeAg levels in HepG2.2.15 cells to a certain extent. LA reduced HBV DNA, HBsAg/HBeAg, and HBcAg levels in the serum/liver tissues of HBV-HDI C57BL/6 mice during the 3-week treatment and suppressed the withdrawal rebound of HBV DNA and HBsAg in the mice serum. LA increased LC3-II protein expression and the number of autolysosomes/autophagosomes and promoted the degradation of sequestosome 1(p62) protein in vitro and in vivo. LA enhanced the co-localization of LC3 protein with autolysosomes, further confirming the ability of LA to induce a complete autophagy. Knockdown of autophagy-related gene (Atg) 7 or 5 in vitro and administration of 3-methyladenine (an autophagic inhibitor) in vivo disabled the inhibitory efficacy of LA on HBV DNA replication, suggesting that the anti-HBV efficacy of LA depended on its ability of inducing autophagy. LA could enhance lysosomal acidification and improve the function of lysosomes by promoting the protein expression of lysosomal-associated membrane protein (LAMP)-1, LAMP-2, and mature cathepsin D, which may contribute to the autophagic induction of LA. LA inhibited the activation of AKT and mammalian target of rapamycin (mTOR) induced by HBV, which was reversed by IGF-1 (an agonist of the PI3K/AKT/mTOR signaling pathway), indicating that LA elicited autophagy through hampering the PI3K/AKT/mTOR signaling pathway. CONCLUSION: We revealed the anti-HBV activity and mechanism of LA in vitro and in vivo. This study facilitates a new understanding of the anti-HBV potent components of S. miltiorrhiza and sheds light on LA for further development as an active constituent or candidate used in the therapy against HBV infection.

Interplay between Selenium, Selenoproteins and HIV-1 Replication in Human CD4 T-Lymphocytes.

The infection of CD4 T-lymphocytes with human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), disrupts cellular homeostasis, increases oxidative stress and interferes with micronutrient metabolism. Viral replication simultaneously increases the demand for micronutrients and causes their loss, as for selenium (Se). In HIV-infected patients, selenium deficiency was associated with a lower CD4 T-cell count and a shorter life expectancy. Selenium has an important role in antioxidant defense, redox signaling and redox homeostasis, and most of these biological activities are mediated by its incorporation in an essential family of redox enzymes, namely the selenoproteins. Here, we have investigated how selenium and selenoproteins interplay with HIV infection in different cellular models of human CD4 T lymphocytes derived from established cell lines (Jurkat and SupT1) and isolated primary CD4 T cells. First, we characterized the expression of the selenoproteome in various human T-cell models and found it tightly regulated by the selenium level of the culture media, which was in agreement with reports from non-immune cells. Then, we showed that selenium had no significant effect on HIV-1 protein production nor on infectivity, but slightly reduced the percentage of infected cells in a Jurkat cell line and isolated primary CD4 T cells. Finally, in response to HIV-1 infection, the selenoproteome was slightly altered.

Simiao Qingwen Baidu decoction inhibits Epstein-Barr virus-induced B lymphoproliferative disease and lytic viral replication.

CONTEXT: Simiao Qingwen Baidu decoction (SQBD), a traditional Chinese medicine prescription, can ameliorate Epstein-Barr virus (EBV) induced disease. However, its mechanism still remains unknown. OBJECTIVE: To detect the mechanism of SQBD in EBV-induced B lymphoproliferative disease in vitro. MATERIALS AND METHODS: Sprague-Dawley (SD) rats (n = 20) were given SQBD (10 mL/kg) by gavage once a day for 7 d. SQBD-containing serum was obtained from abdominal aortic blood of rats, and diluted with medium to obtain 5%, 10% or 20%-medicated serum. SD rats (n = 10) were given normal saline, and normal serum was collected as a control. EBV-transformed B cells (CGM1) were cultured in medium containing 5%, 10% or 20%-medicated serum. CGM1 cells were treated with normal serum as a control. Cell viability and apoptosis were examined. The expression and activity of proteins were assessed. RESULTS: We found that IC50 (83 ± 26.07%, 24 h; 69.88 ± 4.69%, 48 h) of 10% medicated serum was higher than that of 5% (25.47 ± 6.98%, 24 h; 21.62 ± 7.30%, 48 h) and 20%-medicated serum (51 ± 7.25%, 24 h; 56.03 ± 2.56%, 48 h). Moreover, SQBD promoted apoptosis of CGM1 cells by regulating EBV latency proteins expression. SQBD inhibited EBV-induced lytic viral replication. CONCLUSIONS: Our data confirmed that SQBD inhibits EBV-induced B lymphoproliferative disease and lytic viral replication. This work provides a theoretical basis for the mechanism of SQBD in EBV-induced B lymphoproliferative disease, and SQBD may be an effectively therapeutic drug for EBV-induced B lymphoproliferative disease.

Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication.

Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.

Therapeutic opportunities of edible antiviral plants for COVID-19.

The pandemic of Serious Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) that produces corona virus disease (COVID-19) has challenged the entire mankind by rapidly spreading globally in 210 countries affecting over 25 million people and about 1 million deaths worldwide. It continues to spread, afflicting the health system globally. So far there is no remedy for the ailment and the available antiviral regimens have been unsatisfactory for the clinical outcomes and the mode of treatment has been mainly supportive for the prevention of COVID-19-induced morbidity and mortality. From the time immortal the traditional plant-based ethno-medicines have provided the leads for the treatment of infectious diseases. Phytopharmaceuticals have provided potential and less toxic antiviral drugs as compared to conventional modern therapeutics which are associated with severe toxicities. The ethnopharmacological knowledge about plants has provided food supplements and nutraceuticals as a promise for prevention and treatment of the current pandemic. In this review article, we have attempted to comprehend the information about the edible medicinal plant materials with potential antiviral activity specifically against RNA virus which additionally possess property to improve immunity along with external and internal respiration and exhibit anti-inflammatory properties for the prevention and treatment of the disease. This will open an arena for the development of novel nutraceutical herbal formulations as an alternative therapy that can be used for the prevention and treatment of COVID-19.

Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7.

Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNase L in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and interrelationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3' untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) dinucleotides showed no attenuating effect on replication or specific binding to ZAP by IP. However, the bases 3' and 5' of CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP binding, while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG dinucleotides and bound noncompetitively with CpG-enriched RNA, consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP and colocalized with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP's association with larger cellular complexes may mediate the recruitment of OAS3/RNase L, KHNYN, and other RNA degradation pathways.IMPORTANCE We recently discovered that the OAS3/RNase L antiviral pathway is essential for restriction of CpG- and UpA-enriched viruses, in addition to the requirement for zinc finger antiviral protein (ZAP). The current study provides evidence for the specific dinucleotide and wider recognition contexts associated with virus recognition and attenuation. It further documents the association of ZAP and OAS3 and association with stress granules and a wider protein interactome that may mediate antiviral effects in different cellular compartments. The study provides a striking reconceptualization of the pathways associated with this aspect of antiviral defense.

Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

Viruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.

Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease.

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. There is an urgent need to find new therapeutic methods to prevent and control PEDV. Not only is there a shortage of commercial anti-PEDV drugs, but available commercial vaccines fail to protect against highly virulent PEDV variants. We screened an FDA-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of PEDV replication in Vero and IPEC-J2 cells in vitro. Molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of PEDV 3CL protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). The inhibiting effect of tomatidine on 3CL protease was determined using cleavage visualization and FRET assay. Tomatidine-mediated blocking of 3CL protease activity in PEDV-infected cells was examined by western blot detection of the viral polyprotein in PEDV-infected cells. It indicates that tomatidine inhibits PEDV replication mainly by targeting 3CL protease. In addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalo myocarditis virus (EMCV) and seneca virus A (SVA) in vitro. These results may be helpful in developing a new prophylactic and therapeutic strategy against PEDV and other swine disease infections.

Inhibitory effect of simiao qingwen baidu decoction on epstein-barr virus EA, VCA expression and DNA replication in vitro.

This article aims to investigate the role of Simiao Qingwen Baidu Decoction (traditional Chinese medicine) in Epstein-Barr virus (EBV)-induced infectious mononucleosis. Sprague Dawley rats were given Simiao Qingwen Baidu Decoction by gavage, and the medicated serum was collected. EBV-latent infected human Burkitt lymphomas Raji and EBV-transformed marmosets B lymphoblast cell B95-8 were treated with medicated serum. CCK8 assay and flow cytometry were performed to detect cell proliferation and apoptosis. Indirect immunofluorescence assay was performed to analyze EA or VCA positive expression. The copy-number of EBV-DNA and the gene expression were detected by quantitative PCR or quantitative real-time PCR. We found that the medicated serum inhibited proliferation of Raji and B95-8 cells, especially 10 %-medicated serum. The 10 %-medicated serum significantly suppressed EA expression in Raji cells and VCA expression in B95-8 cells. The expression of BZLF1, BRLF1, BMLF1 and EBNA-1 in Raji cells was significantly inhibited by 10 %-medicated serum. 10 %-medicated serum caused a decrease in the copy-number of EBV-DNA in Raji cells. In conclusion, our data imply that Simiao Qingwen Baidu Decoction represses the expression of EA and VCA, and EBV-DNA replication. Thus, our work suggests that Simiao Qingwen Baidu Decoction may play a vital role in anti-EBV.

Switching Host Metabolism as an Approach to Dampen SARS-CoV-2 Infection.

BACKGROUND: COVID-19 pandemic, a global threat, adversely affects all daily lives, altered governmental plans around the world, and urges the development of therapeutics and prophylactics to avoid the expansion of the viral infection. With the recent gradual opening after long lockdown, several recommendations have been placed, with dietary modification as one of the most important approaches that have been appraised. SUMMARY: Here, we are reviewing how changing the host metabolism, particularly changing the host metabolic state from the carbohydrate-dependent glycolytic state to a fat-dependent ketogenic state, may affect viral replication. Furthermore, the impact of intermittent fasting (IF) in triggering metabolic switch along with the impact of supplementation with medium-chain triglycerides (MCTs) such as lauric acid in repressing the envelope formation and viral replication is also addressed. The amalgamation of IF and a ketogenic diet rich in MCTs is thought to work as a prophylactic measure for normal people and adjunct therapy for infected persons. Key Message: A diet regimen of ketogenic breakfast along with supplementation with two doses of lauric acid-rich MCTs at breakfast and lunch times, followed by 8-12-h IF and a dinner rich with fruits and vegetables, could be a potential prophylactic strategy and adjuvant therapy to combat SARS-CoV-2 infections.

Ethanolic extract of Artemisia campestris subsp. glutinosa (Besser) Batt. inhibits HIV-1 replication in vitro through the activity of terpenes and flavonoids on viral entry and NF-κB pathway.

ETHNO-PHARMACOLOGICAL RELEVANCE: The genus Artemisia spp. is well known for its anti-infectious properties and its high content in anti-infectious compounds, like the well-known sweet wormwood (Artemisia annua L.). Another Artemisia species, Artemisia campestris subsp. glutinosa (Besser) Batt., field wormwood, has been traditionally used as medicinal plant in the Mediterranean region. AIM OF THE STUDY: The aim of this study is to investigate the anti-HIV activity of field wormwood, to identify the compounds responsible for this activity and their structure and mechanism of action. MATERIALS AND METHODS: Antiviral activity of isolated compounds and extracts was evaluated in HIV-1 infections of lymphoblastoid cells. We also evaluated the mechanism of action of isolated compounds. Viral entry was studied comparing the inhibitory effect of isolated compounds on wild type HIV-1 and VSV pseudotyped HIV-1. To assess the viral transcriptional effect, plasmids encoding luciferase reporter genes under the control of the whole genome of HIV-1 or NF-κB or Sp1 transcription factors were transfected in the presence of the compounds under evaluation. Finally, antioxidant activity was assessed by quantitation of reduced and total glutathione in treated cell cultures. RESULTS: Ethanolic and aqueous extracts of Artemisia campestris subsp. glutinosa (Besser) Batt. subsp. glutinosa displayed anti-HIV activity in vitro, although ethanolic extract was more powerful (IC50 14.62 µg/mL). Bio-guided ethanolic extract fractionation leads to the isolation and characterization of two terpenes, damsin and canrenone, and four flavonoids, 6, 2', 4'-trimethoxyflavone, acerosin, cardamonin and xanthomicrol. All the isolated compounds inhibited HIV-1 replication in vitro with IC50 values between the middle nanomolar and the low micromolar range. Their anti-HIV mechanism of action is due to the bloking of viral entry and/or transcription inhibition, without correlation with the antioxidant activity, through interference with the cellular transcription factors NF-κB and Sp1, which are targets that are not currently reached by antiretroviral therapy. CONCLUSION: We describe here the anti-HIV activity of field wormwood, Artemisia campestris subsp. glutinosa (Besser) Batt., and the isolation and study of the mechanism of action of two terpenes and four flavonoids, responsible, at least in part, for its activity, through the inhibition of two different cellular targets affecting the HIV replication cycle. The activity of these compounds in cellular targets could explain why plant extracts can be used in the treatment of different diseases. Besides, the presence of several compounds with dual and different mechanisms of action could prove useful in the treatment of HIV-1 infection, since it could aid to overcome drug resistances and simplify drug therapy. This work is a further step in understanding the anti-infectious activity of wormwood species and their use in treating infectious diseases.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

Ribosomal proteins as a possible tool for blocking SARS-COV 2 virus replication for a potential prospective treatment.

Coronavirus disease (COVID-19) is caused by SARS-COV2 and has resulted in more than four million cases globally and the death cases exceeded 300,000. Normally, a range of surviving and propagating host factors must be employed for the completion of the infectious process including RPs. Viral protein biosynthesis involves the interaction of numerous RPs with viral mRNA, proteins which are necessary for viruses replication regulation and infection inside the host cells. Most of these interactions are crucial for virus activation and accumulation. However, only small percentage of these proteins is specifically responsible for host cells protection by triggering the immune pathway against virus. This research proposes RPs extracted from bacillus sp. and yeast as new forum for the advancement of antiviral therapy. Hitherto, antiviral therapy with RPs-involving viral infection has not been widely investigated as critical targets. Also, exploring antiviral strategy based on RPs could be a promising guide for more potential therapeutics.

The role of iron in viral infections.

Crucial cellular processes such as DNA synthesis and the generation of ATP require iron. Viruses depend on iron in order to efficiently replicate within living host cells. Some viruses selectively infect iron - acquiring cells or influence the cellular iron metabolism via Human hemochromatosis protein (HFE) or hepcidin. During infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus (HCV) iron overload is associated with poor prognosis for the patient and enhanced progression of the disease. Recent findings still lack to fully describe the viral interaction with the host iron metabolism during infection. This review summarizes the current knowledge of the viral regulation on the host cell iron metabolism in order to discuss the therapeutic option of iron chelation as a potential and beneficial adjuvant in antiviral therapy.

Wedelolactone inhibits human cytomegalovirus replication by targeting distinct steps of the viral replication cycle.

Wedelolactone (WDL) is a coumestan present in the plants Eclipta prostrata and Wedelia calendulacea which are used for treatment of a multitude of health problems in traditional medicine. It has previously been shown that WDL exerts antiviral activity against human immunodeficiency virus and hepatitis C virus. In this study, we investigated the effect of WDL on lytic human cytomegalovirus (HCMV) infection. We demonstrate a strong interference with HCMV replication as analyzed in multi-round replication settings. A more detailed analysis of the underlying mechanisms revealed that WDL acts at two distinct steps of the viral replication cycle. During immediate early (IE) times, we observe an inhibition of IE1/IE2 expression. Although WDL was reported to interfere with NF-κB signaling our results suggest the existence of additional mechanisms that impede viral IE expression. During later time points of infection, WDL induced a disruption of the interaction between EZH2 and EED, components of the virus-supportive polycomb repressive complex 2 (PRC2). Thereby, the stability of the PRC2 complex as well as the related complex PRC1 was disturbed leading to diminished viral DNA synthesis. Taken together, we identify WDL as a potent agent against HCMV which interferes at two distinct steps of viral replication.

Regulatory aspects of quality and safety for live recombinant viral vaccines against infectious diseases in Japan.

Recombinant viral vaccines expressing antigens of pathogenic microbes (e.g., HIV, Ebola virus, and malaria) have been designed to overcome the insufficient immune responses induced by the conventional vaccines. Our knowledge of and clinical experience with the new recombinant viral vaccines are insufficient, and a clear regulatory pathway is needed for the further development and evaluation of recombinant viral vaccines. In 2018, the research group supported by the Ministry of Health, Labour and Welfare, Japan (MHLW) published a concept paper to address the development of recombinant viral vaccines against infectious diseases. Herein we summarize the concept paper-which explains the Japanese regulatory concerns about recombinant viral vaccines-and provide a focus of discussion about the development of recombinant viral vaccines.

The Combination of Dimorphandra gardneriana Galactomannan and Mangiferin Inhibits Herpes Simplex and Poliovirus.

BACKGROUND: Herpes simplex virus (HSV) and poliovirus (PV) are both agents of major concern in the public health system. It has been shown that Dimorphandra gardneriana galactomannans can be used as solubilizer vehicles in the manufacturing of medicine. Mangiferin is the major constituent of Mangifera indica and presents multiple medicinal and biological activities. OBJECTIVE: This study assayed the effect of D. gardneriana galactomannan combined with mangiferin (DgGmM) against HSV-1 and PV-1. METHODS: The DgGmM cytotoxicity was evaluated by the colorimetric MTT method and the antiviral activity by plaque reduction assay, immunofluorescence and polymerase chain reaction (PCR), in HEp-2 cells. RESULTS: The DgGmM showed a 50% cytotoxic concentration (CC50) > 2000 µg/mL. The 50% inhibitory concentrations (IC50) for HSV-1 and PV-1 were, respectively, 287.5 µg/mL and 206.2 µg/mL, with selectivity indexes (SI) > 6.95 for the former and > 9.69 for the latter. The DgGmM time-ofaddition protocol for HSV-1 showed a maximum inhibition at 500 µg/mL, when added concomitantly to infection and at the time 1 h post-infection (pi). While for PV-1, for the same protocol, the greatest inhibition, was also observed concomitantly to infection at 500 µg/mL and at the times 4 h and 8 h pi. The inhibition was also demonstrated by the decrease of fluorescent cells and/or the inhibition of specific viral genome. CONCLUSION: These results suggested that the DgGmM inhibited HSV-1 and PV-1 replication, with low cytotoxicity and high selectivity and, therefore, represents a potential candidate for further studies on the control of herpes and polio infections.

Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation.

Enterovirus 71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, and no effective treatment is available. Characterization of key host factors is important for understanding its pathogenesis and developing antiviral drugs. Here we report that Hsp27 is one of the most upregulated proteins in response to EV-A71 infection, as revealed by two-dimensional gel electrophoresis-based proteomics studies. Depletion of Hsp27 by small interfering RNA or CRISPR/Cas9-mediated knockout significantly inhibited viral replication, protein expression, and reproduction, while restoration of Hsp27 restored such virus activities. Furthermore, we show that Hsp27 plays a crucial role in regulating viral internal ribosome entry site (IRES) activities by two different mechanisms. Hsp27 markedly promoted 2Apro-mediated eukaryotic initiation factor 4G cleavage, an important process for selecting and initiating IRES-mediated translation. hnRNP A1 is a key IRES trans-acting factor (ITAF) for enhancing IRES-mediated translation. Surprisingly, knockout of Hsp27 differentially blocked hnRNP A1 but not FBP1 translocation from the nucleus to the cytoplasm and therefore abolished the hnRNP A1 interaction with IRES. Most importantly, the Hsp27 inhibitor 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP), a compound isolated from a traditional Chinese herb, significantly protected against cytopathic effects and inhibited EV-A71 infection. Collectively, our results demonstrate new functions of Hsp27 in facilitating virus infection and provide novel options for combating EV-A71 infection by targeting Hsp27.IMPORTANCE Outbreaks of infections with EV-A71, which causes hand, foot, and mouth disease, severe neurological disorders, and even death, have been repeatedly reported worldwide in recent decades and are a great public health problem for which no approved treatments are available. We show that Hsp27, a heat shock protein, supports EV-A71 infection in two distinct ways to promote viral IRES-dependent translation. A small-molecule Hsp27 inhibitor isolated from a traditional Chinese medicinal herb effectively reduces virus yields. Together, our findings demonstrate that Hsp27 plays an important role in EV-A71 infection and may serve as an antiviral target.