ABSTRACT
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the pandemic disease COVID-19, which is so far without efficacious treatment. The discovery of therapy reagents for treating COVID-19 are urgently needed, and the structures of the potential drug-target proteins in the viral life cycle are particularly important. SARS-CoV-2, a member of the Orthocoronavirinae subfamily containing the largest RNA genome, encodes 29 proteins including nonstructural, structural and accessory proteins which are involved in viral adsorption, entry and uncoating, nucleic acid replication and transcription, assembly and release, etc. These proteins individually act as a partner of the replication machinery or involved in forming the complexes with host cellular factors to participate in the essential physiological activities. This review summarizes the representative structures and typically potential therapy agents that target SARS-CoV-2 or some critical proteins for viral pathogenesis, providing insights into the mechanisms underlying viral infection, prevention of infection, and treatment. Indeed, these studies open the door for COVID therapies, leading to ways to prevent and treat COVID-19, especially, treatment of the disease caused by the viral variants are imperative.
Subject(s)
Antiviral Agents/therapeutic use , COVID-19 Drug Treatment , Drug Design/trends , Drug Repositioning , SARS-CoV-2/drug effects , Adrenal Cortex Hormones/chemistry , Adrenal Cortex Hormones/therapeutic use , Antibodies, Viral/chemistry , Antibodies, Viral/therapeutic use , Antiviral Agents/chemistry , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/therapeutic use , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/therapeutic use , Humans , Models, Molecular , Nucleosides/chemistry , Nucleosides/therapeutic use , Protein Conformation , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , Virus Internalization/drug effects , Virus Release/drug effects , Virus Replication/drug effectsABSTRACT
Walleye dermal sarcoma virus (WDSV) is a type of retrovirus, which affects most of the adult walleye fishes during the spawning time. The virus causes multiple epithelial tumors on the fish's skin and fins that are liable for more than 50% of the mortality rate of fish around the world. Till now, no effective antiviral drug or vaccine candidates have been developed that can block the progression of the disease caused by the pathogen. It was found that the 582-amino-acid (aa) residues long internal structural gag polyprotein of the virus plays an important role in virus budding and virion maturation outside of the cell. Inhibition of the protein can block the budding and virion maturation process and can be developed as an antiviral drug candidate against the virus. Therefore, the study aimed to identify potential natural antiviral drug candidates from the tropical mangrove marine plant Avicennia alba, which will be able to block the budding and virion maturation process by inhibiting the activity of the gag protein of the virus. Initially, a homology modeling approach was applied to identify the 3D structure, followed by refinement and validation of the protein. The refined protein structures were then utilized for molecular docking simulation. Eleven phytochemical compounds have been isolated from the marine plant and docked against the virus gag polyprotein. Three compounds, namely Friedlein (CID244297), Phytosterols (CID12303662), and 1-Triacontanol (CID68972) have been selected based on their docking score -8.5 kcal/mol, -8.0 kcal/mol and -7.9 kcal/mol, respectively, and were evaluated through ADME (Absorption, Distribution, Metabolism and Excretion), and toxicity properties. Finally, molecular dynamics (MD) simulation was applied to confirm the binding stability of the protein-ligands complex structure. The ADME and toxicity analysis reveal the efficacy and non-toxic properties of the compounds, where MD simulation confirmed the binding stability of the selected three compounds with the targeted protein. This computational study revealed the virtuous value of the selected three compounds against the targeted gag polyprotein and will be effective and promising antiviral candidates against the pathogen in a significant and worthwhile manner. Although in vitro and in vivo study is required for further evaluation of the compounds against the targeted protein.
Subject(s)
Antiviral Agents/pharmacology , Avicennia/chemistry , Epsilonretrovirus/drug effects , Fish Diseases/prevention & control , Plant Extracts/pharmacology , Retroviridae Infections/veterinary , Tumor Virus Infections/veterinary , Animals , Antiviral Agents/isolation & purification , Epsilonretrovirus/metabolism , Epsilonretrovirus/pathogenicity , Fish Diseases/virology , Gene Products, gag/antagonists & inhibitors , Gene Products, gag/metabolism , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Extracts/isolation & purification , Protein Conformation , Retroviridae Infections/prevention & control , Retroviridae Infections/virology , Structure-Activity Relationship , Tumor Virus Infections/prevention & control , Tumor Virus Infections/virology , Virus Release/drug effectsABSTRACT
Mosquito-borne Zika virus (ZIKV) is a Flavivirus that came under intense study from 2014 to 2016 for its well-known ability to cause congenital microcephaly in fetuses and neurological Guillain-Barré disease in adults. Substantial research on screening antiviral agents against ZIKV and preventing ZIKV infection are globally underway, but Food and Drug Administration (FDA)-approved treatments are not available yet. Compounds from Chinese medicinal herbs may offer an opportunity for potential therapies for anti-ZIKV infection. In this study, we evaluated the antiviral efficacy of harringtonine against ZIKV. Harringtonine possessed anti-ZIKV properties against the binding, entry, replication, and release stage through the virus life cycle. In addition, harringtonine have strong virucidal effects in ZIKV and exhibited prophylaxis antiviral ability prior ZIKV infection. The antiviral activity also observed in the treatment against Japanese encephalitis reporter virus (RP9-GFP strain). Overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-ZIKV infection therapies.
Subject(s)
Antiviral Agents/pharmacology , Harringtonines/pharmacology , Zika Virus Infection/virology , Zika Virus/drug effects , Animals , Antiviral Agents/chemistry , Cells, Cultured , Chlorocebus aethiops , Harringtonines/chemistry , Humans , Models, Molecular , Molecular Conformation , Structure-Activity Relationship , Vero Cells , Viral Envelope Proteins/antagonists & inhibitors , Viral Envelope Proteins/chemistry , Virus Attachment/drug effects , Virus Internalization/drug effects , Virus Release , Virus Replication/drug effects , Zika Virus/genetics , Zika Virus Infection/drug therapyABSTRACT
Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.
Subject(s)
Cell Culture Techniques/methods , Cell Polarity , Hepatocytes/physiology , Induced Pluripotent Stem Cells/physiology , Antiviral Agents/pharmacology , Cell Differentiation , Cells, Cultured , Drug Evaluation, Preclinical/methods , Drug Interactions , Hepatitis A Virus, Human/physiology , Hepatitis E virus/physiology , Hepatocytes/ultrastructure , Hepatocytes/virology , Humans , Liver/cytology , Liver/metabolism , Membrane Transport Proteins/metabolism , Microscopy, Electron, Transmission , Proof of Concept Study , Virion/metabolism , Virus Release , Virus ReplicationABSTRACT
Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.
Subject(s)
Antiviral Agents/pharmacology , Ebolavirus/drug effects , Nucleoproteins/antagonists & inhibitors , Viral Core Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Chlorocebus aethiops , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Ebolavirus/genetics , Gene Expression Regulation, Viral/drug effects , HEK293 Cells , Hemorrhagic Fever, Ebola/drug therapy , Hemorrhagic Fever, Ebola/virology , Humans , Medical Countermeasures , Molecular Structure , Nucleoproteins/chemistry , Pyrimidine Nucleosides/pharmacology , Vero Cells , Viral Core Proteins/chemistry , Virus Release/drug effectsABSTRACT
BACKGROUND: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is a malignant disease in ducklings, causing economic losses in the duck industry. However, there is still no antiviral drug against DHAV-1 in the clinic. OBJECTIVE: Our aim is to investigate the anti-DHAV-1 effect of baicalin, which is a flavonoid derived from the Chinese medicinal herb huangqin (Scutellaria baicalensis Georgi). METHODS: Here, we first detected its anti-DHAV-1 ability in vitro and in vivo. At the same time, the inhibition of baicalin on DHAV-1 reproduction was determined. Finally, we tested and verified the anti-oxidative and immuno-enhancing roles of baicalin on its curative effect on DVH. RESULTS: Baicalin possessed anti-DHAV-1 effect. It improved the cytoactive of DEH which was infected by DHAV-1 as well as reduced the DHAV-1 reproduction in DEH. Under baicalin treatment, mortality of ducklings infected by DHAV-1 decreased, additionally the DHAV-1 level and liver injury in such ducklings were significantly reduced or alleviated. The in vitro mechanism study indicated baicalin inhibited DHAV-1 reproduction via interfering the viral replication and release. Furthermore, the in vivo mechanism study manifested both the anti-oxidative and immuno-enhancing abilities of baicalin, which played crucial roles in its curative effect on DVH. CONCLUSION: This study may provide a scientific basis for developing baicalin as one or a part of the anti-DHAV-1 drugs.
Subject(s)
Antiviral Agents/pharmacology , Flavonoids/pharmacology , Hepatitis Virus, Duck/drug effects , Hepatitis, Viral, Animal/virology , Animals , Antioxidants/pharmacology , Cells, Cultured , Ducks , Hepatitis Virus, Duck/genetics , Hepatitis, Viral, Animal/drug therapy , Hepatitis, Viral, Animal/mortality , Hepatitis, Viral, Animal/pathology , Hepatocytes/drug effects , Hepatocytes/virology , Liver Function Tests , Plant Extracts , Scutellaria baicalensis , Virus Release/drug effects , Virus Replication/drug effectsABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pork industry as it continues to cause significant economic loss worldwide. Currently, vaccination strategies provide very limited protection against PRRSV transmission. Consequently, there is an urgent need to develop new antiviral strategies. Platycodin D (PD) is one of the major bioactive triterpenoid saponins derived from Platycodon grandiflorum, a traditional Chinese medicine used as an expectorant for pulmonary diseases and a remedy for respiratory disorders. Here, we demonstrate that PD exhibits potent activity against PRRSV infection in Marc-145 cells and primary porcine alveolar macrophages. PD exhibited broad-spectrum inhibitory activities in vitro against high pathogenic type 2 PRRSV GD-HD strain and GD-XH strain as well as classical CH-1a and VR2332 strains. PD at concentrations ranging 1â»4 µM significantly inhibited PRRSV RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. EC50 values of PD against four tested PRRSV strains infection in Marc-145 cells ranged from 0.74 to 1.76 µM. Mechanistically, PD inhibited PRRSV replication by directly interacting with virions therefore affecting multiple stages of the virus life cycle, including viral entry and progeny virus release. In addition, PD decreased PRRSV- and LPS-induced cytokine (IFN-α, IFN-ß, IL-1α, IL-6, IL-8 and TNF-α) production in PAMs. Altogether, our findings suggested that PD is a potent inhibitor of PPRSV infection in vitro. However, further in vivo studies are necessary to confirm PD as a potential novel and effective PPRSV inhibitor in swine.
Subject(s)
Antiviral Agents/pharmacology , Porcine respiratory and reproductive syndrome virus/drug effects , Porcine respiratory and reproductive syndrome virus/physiology , Saponins/pharmacology , Triterpenes/pharmacology , Virus Replication/drug effects , Animals , Cells, Cultured , Microbial Sensitivity Tests , Swine , Virus Internalization/drug effects , Virus Release/drug effectsABSTRACT
Hepatitis C virus (HCV) infection is a worldwide public health burden and it is estimated that 185 million people are or have previously been infected worldwide. There is no effective vaccine for prevention of HCV infection; however, a number of drugs are available for the treatment of infection. The availability of direct-acting antivirals (DAAs) has dramatically improved therapeutic options for HCV genotype 1. However, the high costs and potential for development of resistance presented by existing treatment demonstrate the need for the development of more efficient new antivirals, or combination of therapies that target different stages of the viral lifecycle. Over the past decades, there has been substantial study of compounds extracted from plants that have activity against a range of microorganisms that cause human diseases. An extensive variety of natural compounds has demonstrated antiviral action worldwide, including anti-HCV activity. In this context, plant-derived compounds can provide an alternative approach to new antivirals. In this review, we aim to summarize the most promising plant-derived compounds described to have antiviral activity against HCV.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Plant Extracts/pharmacology , Antiviral Agents/chemistry , Biological Products/chemistry , Biological Products/pharmacology , Cell Line , Cells, Cultured , Hepatitis C/virology , Humans , Plant Extracts/chemistry , Structure-Activity Relationship , Virus Assembly/drug effects , Virus Internalization/drug effects , Virus Release/drug effects , Virus Replication/drug effectsABSTRACT
Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.
Subject(s)
Hemagglutinins/metabolism , Neuraminidase/metabolism , Orthomyxoviridae/metabolism , Plant Extracts/metabolism , Taxodium/chemistry , Animals , Cell Line , Cell Survival/drug effects , Dogs , Hemagglutinins/chemistry , Humans , Madin Darby Canine Kidney Cells , Microscopy, Fluorescence , Neuraminidase/antagonists & inhibitors , Orthomyxoviridae/enzymology , Plant Extracts/chemistry , Plant Extracts/toxicity , RNA, Viral/chemistry , RNA, Viral/metabolism , Taxodium/metabolism , Viral Proteins/metabolism , Virus Internalization/drug effects , Virus Release/drug effectsABSTRACT
Subcellular trafficking within host cells plays a critical role in viral life cycles, including influenza A virus (IAV). Thus targeting relevant subcellular compartments holds promise for effective intervention to control the impact of influenza infection. Bafilomycin A1 (Baf-A1), when used at relative high concentrations (≥10 nM), inhibits vacuolar ATPase (V-ATPase) and reduces endosome acidification and lysosome number, thus inhibiting IAV replication but promoting host cell cytotoxicity. We tested the hypothesis that much lower doses of Baf-A1 also have anti-IAV activity, but without toxic effects. Thus we assessed the antiviral activity of Baf-A1 at different concentrations (0.1-100 nM) in human alveolar epithelial cells (A549) infected with IAV strain A/PR/8/34 virus (H1N1). Infected and mock-infected cells pre- and cotreated with Baf-A1 were harvested 0-24 h postinfection and analyzed by immunoblotting, immunofluorescence, and confocal and electron microscopy. We found that Baf-A1 had disparate concentration-dependent effects on subcellular organelles and suppressed affected IAV replication. At concentrations ≥10 nM Baf-A1 inhibited acid lysosome formation, which resulted in greatly reduced IAV replication and release. Notably, at a very low concentration of 0.1 nM that is insufficient to reduce lysosome number, Baf-A1 retained the capacity to significantly impair IAV nuclear accumulation as well as IAV replication and release. In contrast to the effects of high concentrations of Baf-A1, very low concentrations did not exhibit cytotoxic effects or induce apoptotic cell death, based on morphological and FACS analyses. In conclusion, our results reveal that low-concentration Baf-A1 is an effective inhibitor of IAV replication, without impacting host cell viability.
Subject(s)
Alveolar Epithelial Cells/virology , Antiviral Agents/pharmacology , Influenza A Virus, H1N1 Subtype/physiology , Macrolides/pharmacology , Virus Replication/drug effects , Animals , Autophagy , Cell Line, Tumor , Dogs , Drug Evaluation, Preclinical , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Madin Darby Canine Kidney Cells , Virus Attachment , Virus Release/drug effectsABSTRACT
Scutellaria baicalensis Georgi, a Chinese herbal decoction, has been used for the treatment of the common cold, fever and influenza virus infections. In previous studies, we found that oral administration of baicalein resulted in the inhibition of influenza A virus replication in vivo, which was linked to baicalin in serum. However, the effective dose and underlying mechanisms of the efficacy of baicalin against influenza A virus have not been fully elucidated. In this study, the antiviral effects of baicalin in influenza-virus-infected MDCK cells and mice were examined. The neuraminidase inhibition assay was performed to investigate the mechanism of action of baicalin. In vitro results showed that baicalin exhibited a half-maximal effective concentration (EC50) of 43.3 µg/ml against the influenza A/FM1/1/47 (H1N1) virus and 104.9 µg/ml against the influenza A/Beijing/32/92 (H3N2) virus. When added to MDCK cell cultures after inoculation with influenza virus, baicalin demonstrated obvious antiviral activity that increased in a dose-dependent manner, indicating that baicalin affected virus budding. Baicalin had clear inhibitory effects against neuraminidases, with half-maximal inhibitory concentration (IC50) of 52.3 µg/ml against the influenza A/FM1/1/47 (H1N1) virus and 85.8 µg/ml against the influenza A/Beijing/32/92 (H3N2) virus. In vivo studies showed that an intravenous injection of baicalin effectively reduced the death rate, prolonged the mean day to death (MDD) and improved the lung parameters of mice infected with influenza A virus. These results demonstrate that baicalin acts as a neuraminidase inhibitor, with clear inhibitory activities that are effective against different strains of influenza A virus in both cell culture and a mouse model, and that baicalin has potential utility in the management of influenza virus infections.
Subject(s)
Antiviral Agents/pharmacology , Flavonoids/pharmacology , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/drug effects , Neuraminidase/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Administration, Intravenous , Animals , Antiviral Agents/isolation & purification , Antiviral Agents/therapeutic use , Cell Culture Techniques , Disease Models, Animal , Dogs , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Flavonoids/isolation & purification , Flavonoids/therapeutic use , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H3N2 Subtype/enzymology , Influenza A Virus, H3N2 Subtype/physiology , Inhibitory Concentration 50 , Lung/pathology , Lung/virology , Madin Darby Canine Kidney Cells , Mice , Microbial Sensitivity Tests , Orthomyxoviridae Infections/drug therapy , Scutellaria/chemistry , Survival Analysis , Treatment Outcome , Virus Release/drug effectsABSTRACT
Various drugs found in Chinese herbs are well known for their antiviral potency. We have tested several flavonoids with respect to their potency to block the viral protein U of the human immunodeficiency type 1 virus, which is believed to form a cation-permeable ion channel in the infected cell. We used Xenopus oocytes with heterologously expressed viral protein U as model system to test the efficacy of the drugs in voltage-clamp experiments. This method had been demonstrated in the past as a useful tool to screen drugs for their potency in inhibition of ion channel activity. The viral protein U-mediated current could be inhibited by Ba(2+) with a K1/2 value of 1.6 mM. Therefore, we determined viral protein U-mediated current as current component blocked by 10 mM Ba(2+). We screened several flavonoids with respect to their effects on this current. The flavonols quercetin and kaempferol, and the flavanols (-)epigallochatechin and (-)epichatechin were ineffective. The flavanone naringenin showed at 20 µM slight (about 10%) inhibition. The most potent drug was the isoflavon genistein which exhibited at 20 µM significant inhibition of about 40% with a K1/2 value of 81 ± 4 µM. We suggest that viral ion channels, in general, may be a good target for development of antiviral agents, and that, in particular, isoflavons may be candidates for development of drugs targeting viral protein U.
Subject(s)
Antiviral Agents/pharmacology , Flavanones/pharmacology , Flavonoids/pharmacology , Genistein/pharmacology , HIV Infections/drug therapy , HIV-1/drug effects , Animals , Antiviral Agents/chemistry , Female , Flavanones/chemistry , Flavonoids/chemistry , Gene Expression , Genistein/chemistry , Human Immunodeficiency Virus Proteins/antagonists & inhibitors , Humans , Ion Channels/drug effects , Patch-Clamp Techniques , RNA, Complementary/genetics , Transgenes , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Virus Release/drug effects , Xenopus laevisABSTRACT
Phage therapy presents an alternative therapeutic option in treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains not responding to antibiotic therapy. However, it is essential to study the role of external factors that may influence the yield and potency of phage preparations intended for use in various in vitro and in vivo studies. The present study focuses on the effects of calcium in the entire infection process of a broad-spectrum lytic bacteriophage: MR-10. The presence of calcium increased the adsorption rate of the phage and also participated in the process of penetration of the phage genome into the host cytoplasm. A final concentration of 5 mM of calcium ions supplemented in soft agar during the phage titration process significantly increased the phage titer. Hence, incorporation of such divalent cations during the isolation of lytic phages active against MRSA strains and during the preparation of high-titer active phage preparations would definitely increase the isolation frequency and the final phage yield. This will contribute towards more effective phage preparations for use in treatments against MRSA infections.
Subject(s)
Calcium/pharmacology , Methicillin-Resistant Staphylococcus aureus/virology , Virus Attachment/drug effects , Virus Internalization/drug effects , Virus Release/drug effects , Bacteriophages/pathogenicity , Staphylococcal Infections/therapyABSTRACT
Carthamus tinctorius L. (CT) is traditionally used to reduce ailments from diseases of the musculoskeletal system and connective tissue and diseases of blood circulation and the cardiovascular system. Flower extracts from CT are known to have antibacterial activity, anti-inflammatory activity, and to inhibit tumor promotion in mouse skin carcinogenesis. In order to discover new antiviral agents from CT extracts, we tested whether CT extracts contain antiviral activity against gammaherpesvirus infection. This study demonstrated that treatment with CT extracts disrupted KSHV latency in the viral-infected host cells, iSLK-BAC16. n-Hexane and EtOH fractions of CT extracts critically affected at least two stages of the KHSV life-cycle by abnormally inducing KSHV lytic reactivation and by severely preventing KSHV virion release from the viral host cells. In addition to the effects on KSHV itself, CT extract treatments induced cellular modifications by dysregulating cell-cycle and producing strong cytotoxicity. This study demonstrated for the first time that CT extracts have antiviral activities that could be applied to development of new anti-gammaherpesviral agents.
Subject(s)
Antiviral Agents/pharmacology , Carthamus tinctorius/chemistry , Herpesvirus 8, Human/drug effects , Herpesvirus 8, Human/physiology , Plant Extracts/pharmacology , Virus Replication/drug effects , Animals , Antiviral Agents/toxicity , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line , Chlorocebus aethiops , Gene Expression Regulation, Viral/drug effects , Humans , Plant Extracts/toxicity , Transcription, Genetic , Vero Cells , Virus Activation/drug effects , Virus Latency/drug effects , Virus Release/drug effectsABSTRACT
Concerns about the possible use of variola virus as a biological weapon as well as the need for therapeutics for the treatment or prevention of naturally acquired poxvirus infections or vaccination complications have led to the search for small molecule inhibitors of poxvirus replication. One unique and attractive target for antiviral development is viral egress. Part of understanding the mechanism of action of viral egress inhibitors involves determining which virion form is being made. This can be accomplished through buoyant density centrifugation.
Subject(s)
Antiviral Agents/pharmacology , Benzamides/pharmacology , Isoindoles/pharmacology , Vaccinia virus/drug effects , Virus Release/drug effects , Animals , Blotting, Western , Cell Line , Centrifugation, Density Gradient , Drug Evaluation, Preclinical/methods , Rabbits , Vaccinia virus/isolation & purification , Vaccinia virus/physiology , Viral Proteins/metabolism , Virion/drug effects , Virus Cultivation , Virus Replication/drug effectsABSTRACT
Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.
Subject(s)
Golgi Apparatus/metabolism , Hepacivirus/metabolism , Hepatitis C/metabolism , Membrane Proteins/metabolism , Phosphatidylinositol Phosphates/metabolism , Virus Release/physiology , Biological Transport, Active/genetics , Cell Line, Tumor , Golgi Apparatus/genetics , Golgi Apparatus/virology , Hepacivirus/genetics , Hepatitis C/genetics , Humans , Intracellular Membranes/metabolism , Intracellular Membranes/virology , Membrane Proteins/genetics , Myosins/genetics , Myosins/metabolism , Phosphatidylinositol Phosphates/genetics , Virion/genetics , Virion/metabolismABSTRACT
Certain viral protein-protein interactions provide attractive targets for antiviral drug development. Recently, we described a ß-lactamase based protein fragment complementation assay (PCA) to study the core interaction of the nuclear egress complex (NEC) of different herpesviruses in cells. Now, to have a cell free assay for inhibitor screens, we expressed split ß-lactamase tagged interaction domains of the viral pUL50 and pUL53 proteins representing the NEC of human cytomegalovirus (HCMV) in bacteria. After validation and basic characterization of this NEC-PCA, we tested peptide inhibitors of the pUL50-pUL53 complex. We show that peptides resembling sequences of the first conserved region of pUL53 can inhibit the NEC-PCA. This, on one hand, indicated that the core interaction in the HCMV NEC is mediated by a linear motif. On the other hand it proved that this new pUL50-pUL53 interaction assay allows a simple cell free test for small molecular inhibitors.
Subject(s)
Cytomegalovirus/physiology , Viral Proteins/metabolism , Virology/methods , Virus Release , Antiviral Agents/pharmacology , Bacteria , Drug Evaluation, Preclinical/methods , Humans , Protein Binding , beta-Lactamases/metabolismABSTRACT
BACKGROUND: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. RESULTS: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. CONCLUSIONS: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.
Subject(s)
Biological Therapy , Staphylococcal Infections/microbiology , Staphylococcal Infections/therapy , Staphylococcus Phages/physiology , Staphylococcus aureus/virology , Virus Release , Animals , Endopeptidases/genetics , Endopeptidases/metabolism , Humans , Male , Mice , Staphylococcus Phages/enzymology , Staphylococcus Phages/genetics , Staphylococcus aureus/physiology , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
AIMS: To investigate the in vitro antiherpes effects of the crude aqueous extract obtained from Cecropia glaziovii leaves and their related fractions, the n-butanol fraction (n-BuOH) and the C-glycosylflavonoid-enriched fraction (MeOH(AMB)), and to determine the viral multiplication step(s) upon which this C-glycosylflavonoid-enriched fraction acts. METHODS AND RESULTS: The antiviral activity was evaluated against human herpes virus types 1 and 2 (HHV-1, HHV-2) by plaque reduction assay. The mode of action of the most active fraction was investigated by a set of assays, and the results demonstrated that MeOH(AMB) fraction exerts anti-herpes action by the reduction of viral infectivity (only against HHV-2); by the inhibition of virus entry into cells; by the inhibition of cell-to-cell virus spread as well as by the impaired levels of envelope proteins of HHV-1. The high-performance liquid chromatography (HPLC)-photo-diode array (PDA) analysis showed that the C-glycosylflavonoids are the major constituents of this fraction. CONCLUSIONS: These data showed that the MeOH(AMB) fraction has an antiviral activity against HHV types 1 and 2. The C-glycosylflavonoids are the major constituents of this fraction, which suggests that they could be one of the compounds responsible for the detected anti-herpes activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The MeOH(AMB) fraction can be regarded as a phytopharmaceutical candidate for the treatment of herpetic infections.