Pathway analysis of host responses to dengue virus serotype 2 infection and inhibition of viral envelope protein by naringenin from Ganoderma lucidum.

Dengue fever cases are spiking over the last two decades. Incessant efforts are still being made to gain deeper insights on this arboviral disease and to identify bioactive antivirals. In this study, bioinformatics analysis was conducted to identify the differentially expressed genes (DEGs) in the expression profiling datasets of dengue virus serotype 2 (DENV2) patients. We found overexpressed genes in dengue patients that can interrupt cell cycle progression and phase transitions of mitosis inside the host to favour the viral replication process. These DEGs were associated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as cell cycle and DNA replication. A protein interaction network consisting of these significant pathways was also constructed using STRING. Futher, the traditional Chinese medicine (TCM) compounds from Ganoderma lucidum were screened to target DENV2 envelope protein, which was crucial for viral fusion activity. Docking, orbital energy, and toxicity prediction analysis revealed that naringenin was the best antiviral candidate. Following molecular dynamics simulations, the predicted binding energy of the protein-naringenin system using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach was slightly greater than the control system. It is recommended to perform in vitro inhibition of naringenin against DENV2 and use our findings to complement the experimental data obtained.

Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity.

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

Harringtonine Inhibits Zika Virus Infection through Multiple Mechanisms.

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.

Discovery and Structural Optimization of 4-(Aminomethyl)benzamides as Potent Entry Inhibitors of Ebola and Marburg Virus Infections.

The recent Ebola epidemics in West Africa underscore the great need for effective and practical therapies for future Ebola virus outbreaks. We have discovered a new series of remarkably potent small molecule inhibitors of Ebola virus entry. These 4-(aminomethyl)benzamide-based inhibitors are also effective against Marburg virus. Synthetic routes to these compounds allowed for the preparation of a wide variety of structures, including a conformationally restrained subset of indolines (compounds 41-50). Compounds 20, 23, 32, 33, and 35 are superior inhibitors of Ebola (Mayinga) and Marburg (Angola) infectious viruses. Representative compounds (20, 32, and 35) have shown good metabolic stability in plasma and liver microsomes (rat and human), and 32 did not inhibit CYP3A4 nor CYP2C9. These 4-(aminomethyl)benzamides are suitable for further optimization as inhibitors of filovirus entry, with the potential to be developed as therapeutic agents for the treatment and control of Ebola virus infections.

A simple and rapid pipeline for identification of receptor-binding sites on the surface proteins of pathogens.

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.

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

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

Brain-targeted delivery of PEGylated nano-bacitracin A against Penicillin-sensitive and -resistant Pneumococcal meningitis: formulated with RVG29 and Pluronic® P85 unimers.

Pneumococcal meningitis (PM), caused by Streptococcus pneumonia, remains a high-burden disease in developing countries. Antibiotic therapy has been limited due to the inefficiency of drug transport across the blood-brain barrier (BBB) and the emergence of drug-resistant strains. In our preliminary study, PEGylated nano-self-assemblies of bacitracin A (PEGylated Nano-BA12K) demonstrated a strong antibacterial potency against S. pneumonia. In this study, the potential application of this micelle for the treatment of both Penicillin-sensitive and -resistant PM was studied. To address BBB-targeting and -crossing issues, PEGylated Nano-BA12K was formulated with a specific brain-targeting peptide (rabies virus glycopeptide-29, RVG29) and a P-glycoprotein inhibitor (Pluronic® P85 unimers) to construct a mixed micellar system (RVG29-Nano-BAP85). RVG29-Nano-BAP85 demonstrated a strong antibacterial potency against 13 clinical isolates of S. pneumonia, even higher than that of Penicillin G, a conventional anti-PM agent. RVG29-Nano-BAP85 had more cellular uptake in brain capillary endothelial cells (BCECs) and higher BBB-crossing efficiency than single formulated Nano-BAs as shown in an in vitro BBB model. The enhanced BBB-permeability was attributed to the synergetic effect of RVG29 and P85 unimers through receptor-mediated transcytosis, exhaustion of ATP, and reduction in membrane microviscosity. In vivo results further demonstrated that RVG29-Nano-BAP85 was able to accumulate in brain parenchyma as confirmed by in vivo optical imaging. In addition, RVG29-Nano-BAP85 exhibited high therapeutic efficiencies in both Penicillin-sensitive and -resistant PM mouse models with negligible systemic toxicity. Collectively, RVG29-Nano-BAP85 could effectively overcome BBB barriers and suppressed the growth of both drug-sensitive and -resistant S. pneumonia in the brain tissues, which demonstrated its potential for the treatment of PM.

Discovery of Potent Inhibitors for the Inhibition of Dengue Envelope Protein: An In Silico Approach.

BACKGROUND: Dengue fever, a major public health problem in the tropical and sub-tropical countries caused by the infection of Dengue virus transmitted by the anthropod vectors. The dengue virus infection is represented as the "Neglected Tropical Diseases" by the world health organization. The structural protein E binds to the receptor on the host cell surface during infection and the binding directs to the endocytic pathway. The conformational change of the envelope protein helps to infuse the viral lipid membrane and delivers the viral genome into the cytosol. No specific treatments are available till date and development of the vaccine for the DENV is challenging due to the immunization and longlasting protection against all four serotypes. Hence, identification of potent inhibitors would overlay the therapeutics against the mediated diseases. OBJECTIVE: Our study focuses on developing the novel potent inhibitors to inhibit the viral attachment and membrane fusion of the Dengue virus Envelope protein. METHODS: The crystal structure of Dengue Envelope protein has been retrieved from the protein data bank and optimized through Schrödinger. The structure-based virtual screening based on the cocrystallised ligand has been carried out with the small molecule libraries, and based on the docking score, interaction and energy value best complexes were selected. The selected complexes were further taken forward for the conformational stability analysis through Molecular dynamics simulation. RESULTS: Around 55 molecules from the three databases were identified as potential binders to the envelope protein and the docking studies revealed that the top compounds possess strong interaction with the good energies. The Molecular electrostatic surface potential of the top five compounds states that the interactions were observed mostly in the electropositive region. Finally, the best 5 compounds carried further for molecular dynamics simulations exposed that they were highly stable and no loss of interactions was observed between those complexes. CONCLUSION: Hence, from the results, it is evident that the compounds DB00179, Quercetin, Silymarin, Dapagliflozlin and Fisetin could be novel and potent candidates to inhibit the DENV envelope protein.

Structure based virtual screening of the Ebola virus trimeric glycoprotein using consensus scoring.

Ebola virus (EBOV) causes zoonotic viral infection with a potential risk of global spread and a highly fatal effect on humans. Till date, no drug has gotten market approval for the treatment of Ebola virus disease (EVD), and this perhaps allows the use of both experimental and computational approaches in the antiviral drug discovery process. The main target of potential vaccines that are recently undergoing clinical trials is trimeric glycoprotein (GP) of the EBOV and its exact crystal structure was used in this structure based virtual screening study, with the aid of consensus scoring to select three possible hit compounds from about 36 million compounds in MCULE's database. Amongst these three compounds, (5R)-5-[[5-(4-chlorophenyl)-1,2,4-oxadiazol-3-yl]methyl]-N-[(4-methoxyphenyl)methyl]-4,5-dihydroisoxazole-3-carboxamide (SC-2, C21H19ClN4O4) showed good features with respect to drug likeness, ligand efficiency metrics, solubility, absorption and distribution properties and non-carcinogenicity to emerge as the most promising compound that can be optimized to lead compound against the GP EBOV. The binding mode showed that SC-2 is well embedded within the trimeric chains of the GP EBOV with molecular interactions with some amino acids. The SC-2 hit compound, upon its optimization to lead, might be a good potential candidate with efficacy against the EBOV pathogen and subsequently receive necessary approval to be used as antiviral drug for the treatment of EVD.

Epigallocatechin gallate, an active green tea compound inhibits the Zika virus entry into host cells via binding the envelope protein.

Emerging infections of Zika virus (ZIKV) are associated with serious consequences like microcephaly and Guillain-Barré syndrome. It leads to a situation of global health emergency and demand an intensive research investigation to develop safe and effective therapeutics. Various efforts have been made to reduce the pathological pressure of ZIKV, but no effective drug has been introduced against ZIKV infections. A recent study has reported the inhibition of ZIKV entry into the host cells by an active green tea ingredient, Epigallocatechin Gallate (EGCG) in Vero E6cells. The effect of EGCG seems remarkable but lacking the information of the mechanism of action. In this study, we have investigated the binding site (Site1) of EGCG on envelope protein and provided the insights into various interactions of molecule with the binding site using molecular docking studies. Further, using molecular dynamics approaches we proposed the possible associated mechanism of inhibition of ZIKV entry by EGCG molecule. EGCG has found to interact with several residues and providing stability to the protein conformations up to 50ns simulations.

Targeting the dengue ß-OG with serotype-specific alkaloid virtual leads.

The dengue envelope ß-OG pocket is a crucial hinge for mediating virus-host fusion via conformational changes in the envelope to the fusion-competent form. The ß-OG pocket is a small molecule target site for inhibition of virus-host fusion. As of date, the only structure of the ß-OG pocket known is of serotype 2. Studies of ß-OG inhibition by small molecules primarily target viral serotype 2. Envelope and ß-OG sequence alignments, reveal dissimilarities across serotypes. In light of protein sequence-structure-function correlation, sequence variations suggest serotypic variations in ß-OG druggability. This, together with the fact that dengue viral proteins do have serotype-specific variations of structure and function, lead to the study of the serotype-specificity of the dengue ß-OG ligand binding behaviour. ß-OG druggability was compared using comparative models of envelope proteins containing the ß-OG pocket in four serotypes of the dengue virus. ß-OG ligand binding was found to vary with respect to hydrophobicity, hydrophilicity, hydrogen bonding, van der Waals interactions with ligands and tightness of the binding site. The study also reports serotype-specific virtual leads identified from a library of 9175 alkaloids, using a consensus docking and scoring approach. The docking algorithms of Glide SP and XP, together with the Lamarckian genetic algorithm were employed for consensus docking. For consensus scoring, the Glide empirical score was employed along with the scoring function of AutoDock. A multi-dimensional lead optimisation approach was performed for optimising affinity, ligand efficiency, lipophilic ligand efficiency, ADMET and molecular torsional strains. The study proposes the serotype-specific inhibition of the ß-OG for an effective inhibition of virus-host fusion, in contrast to a pan inhibitor.

Casamino acids facilitate the secretion of recombinant dengue virus serotype-3 envelope domain III in Pichia pastoris.

BACKGROUND: Dengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of dengue viruses (DENV) that places ~40 % of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope domain-III (EDIII) antigen of dengue virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose. RESULTS: In this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20 °C than 30 °C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20 °C in the presence of 1 % CA. The overall increase in EDIII titer was ~9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV. CONCLUSIONS: The strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement.

In silico assessment of phosphorylation and O-ß-GlcNAcylation sites in human NPC1 protein critical for Ebola virus entry.

Ebola is a highly pathogenic enveloped virus responsible for deadly outbreaks of severe hemorrhagic fever. It enters human cells by binding a multifunctional cholesterol transporter Niemann-Pick C1 (NPC1) protein. Post translational modification (PTM) information for NPC1 is crucial to understand Ebola virus (EBOV) entry and action due to changes in phosphorylation or glycosylation at the binding site. It is difficult and costly to experimentally assess this type of interaction, so in silico strategy was employed. Identification of phosphorylation sites, including conserved residues that could be possible targets for 21 predicted kinases was followed by interplay study between phosphorylation and O-ß-GlcNAc modification of NPC1. Results revealed that only 4 out of 48 predicted phosphosites exhibited O-ß-GlcNAc activity. Predicted outcomes were integrated with residue conservation and 3D structural information. Three Yin Yang sites were located in the α-helix regions and were conserved in studied vertebrate and mammalian species. Only one modification site S425 was found in ß-turn region located near the N-terminus of NPC1 and was found to differ in pig, mouse, cobra and humans. The predictions suggest that Yin Yang sites may not be important for virus attachment to NPC1, whereas phosphosite 473 may be important for binding and hence entry of Ebola virus. This information could be useful in addressing further experimental studies and therapeutic strategies targeting PTM events in EBOV entry.

Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate.

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes.

In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles.

Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 µg Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 µg dose of E2 adsorbed to 250 µg HMSA was compared to immunisation with Opti-E2 (50 µg) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 µg). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.

Computational repositioning of ethno medicine elucidated gB-gH-gL complex as novel anti herpes drug target.

BACKGROUND: Herpes viruses are important human pathogens that can cause mild to severe lifelong infections with high morbidity. They remain latent in the host cells and can cause recurrent infections that might prove fatal. These viruses are known to infect the host cells by causing the fusion of viral and host cell membrane proteins. Fusion is achieved with the help of conserved fusion machinery components, glycoproteins gB, heterodimer gH-gL complex along with other non-conserved components. Whereas, another important glycoprotein gD without which viral entry to the cell is not possible, acts as a co-activator for the gB-gH-gL complex formation. Thus, this complex formation interface is the most promising drug target for the development of novel anti-herpes drug candidates. In the present study, we propose a model for binding of gH-gL to gB glycoprotein leading from pre to post conformational changes during gB-gH-gL complex formation and reported the key residues involved in this binding activity along with possible binding site locations. To validate the drug targetability of our proposed binding site, we have repositioned some of the most promising in vitro, in vivo validated anti-herpes molecules onto the proposed binding site of gH-gL complex in a computational approach. METHODS: Hex 6.3 standalone software was used for protein-protein docking studies. Arguslab 4.0.1 and Accelrys® Discovery Studio 3.1 Visualizer softwares were used for semi-flexible docking studies and visualizing the interactions respectively. Protein receptors and ethno compounds were retrieved from Protein Data Bank (PDB) and Pubchem databases respectively. Lipinski's Filter, Osiris Property Explorer and Lazar online servers were used to check the pharmaceutical fidelity of the drug candidates. RESULTS: Through protein-protein docking studies, it was identified that the amino acid residues VAL342, GLU347, SER349, TYR355, SER388, ASN395, HIS398 and ALA387 of gH-gL complex play an active role in its binding activity with gB. Semi flexible docking analysis of the most promising in vitro, in vivo validated anti-herpes molecules targeting the above mentioned key residues of gH-gL complex showed that all the analyzed ethno medicinal compounds have successfully docked into the proposed binding site of gH-gL glycoprotein with binding energy range between -10.4 to -6.4 K.cal./mol. CONCLUSIONS: Successful repositioning of the analyzed compounds onto the proposed binding site confirms the drug targetability of gH-gL complex. Based on the free binding energy and pharmacological properties, we propose (3-chloro phenyl) methyl-3,4,5 trihydroxybenzoate as worth a small ethno medicinal lead molecule for further development as potent anti-herpes drug candidate targeting gB-gH-gL complex formation interface.

Effect of envelope proteins on the mechanical properties of influenza virus.

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm(-1)) than a viral protein-capsid coat and sustains deformations of one-third of the virion's diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.

Contortrostatin, a homodimeric disintegrin isolated from snake venom inhibits herpes simplex virus entry and cell fusion.

BACKGROUND: Herpes simplex virus (HSV) causes significant health problems from periodical skin and corneal lesions to encephalitis. HSV entry provides a unique opportunity for therapeutic intervention. In this study, we evaluated contortrostatin (CN), an Arg-Gly-Asp motif containing disintegrin isolated from snake venom, as a novel therapeutic agent with ability to block HSV entry and related membrane fusion. METHODS: In vitro efficacy of CN against HSV was determined using an HSV type-1 (HSV-1) entry assay based on the measurement of ß-galactosidase reporter activity originating from the genome of a recombinant strain of HSV-1(KOS) gL86. HSV-1 glycoprotein-mediated cell-to-cell fusion was used to study the effect of CN on polykaryocyte formation. Primary as well as transformed cell lines were used for this study. RESULTS: Pre-treatment of Chinese hamster ovary (CHO-K1) cells expressing HSV-1 glycoprotein D receptors and primary cultures of human corneal fibroblasts (CF) with CN resulted in the inhibition of entry, cell-to-cell fusion, and polykaryocyte formation. Interestingly, a more pronounced anti-HSV-1 effect was observed in naturally susceptible CF than CHO-K1 cells. CONCLUSIONS: CN, a novel venom-derived peptide, exhibits the ability to block two key steps, entry and cell-to-cell fusion, in HSV infection. Showing strong promise for development as an anti-HSV agent, it also demonstrates better prophylactic efficacy in primary cells.

Proanthocyanidin-enriched extract from Myrothamnus flabellifolia Welw. exerts antiviral activity against herpes simplex virus type 1 by inhibition of viral adsorption and penetration.

AIM OF THE STUDY: Extracts from the aerial parts of the South African resurrection plant Myrothamnus flabellifolia Welw. have been used traditionally against infections of the upper respiratory tract and skin diseases. A polyphenol-enriched extract was investigated for potential antiviral effects against herpes simplex virus type 1 (HSV-1) and adenovirus, and the underlying mode of action was to be studied. MATERIALS AND METHODS: Antiviral effects of an acetone-water extract (MF) from Myrothamnus flabellifolia on HSV-1 and adenovirus type 3 were tested in infected Vero cells by plaque reduction assay, MTT test and immunofluorescence. The influence of the extract on the HSV-1 envelope glycoprotein D was shown by Western blot. Organotypic full thickness skin models consisting of multilayer skin equivalents were used for the investigation of MF effects on HSV-1 replication. RESULTS: MF exhibited strong antiviral activity against HSV-1. The HSV-1-specific inhibitory concentration (IC(50)) was determined as 0.4 µg/mL and the cytotoxic concentration (CC(50)) against Vero cells as 50 µg/mL. A selectivity index (SI) (ratio of CC(50) to IC(50)) of approximately 120 was calculated when MF was added to the virus inoculum for 1h at 37°C prior to infection. The replication of adenovirus 3 was not affected by MF. MF abolished virus entry into the host cell by blocking viral attachment to the cell surface. When added after attachment at a concentration of >6 µg/mL, the extract also inhibited penetration of HSV-1 into the host cell. Polyphenolic compounds from MF directly interacted with viral particles, leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein D (gD). Using organotypic full thickness tissue cultures, it was shown that treatment of HSV-1 infected cultures with the MF resulted in reduced viral spread. CONCLUSIONS: A polyphenol-enriched extract from Myrothamnus flabellifolia strongly acts against HSV-1 by blocking viral entry into the cells.

Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir.

Raltegravir is a recently, Food and Drug Administration-approved, small-molecule drug that inhibits retroviral integrase, thereby preventing HIV DNA from inserting itself into the human genome. We report here that the activity profile of raltegravir on the replication of murine leukemia virus is similar to that for HIV, and that the drug specifically affects autoimmune disease in mice, in which endogenous retroelements are suspected to play a role. While NZW and BALB/c mice, which do not succumb to autoimmune disease, are not affected by raltegravir, lupus-prone (NZBxNZW) F(1) mice die of glomerulonephritis more than a month earlier than untreated mice. Raltegravir-treated NZB mice, which share the H-2 haplotype with BALB/c mice, but which are predisposed to autoimmune hemolytic anemia, develop auto-antibodies to their red blood cells >3 months earlier than untreated mice of the same strain. Because nonautoimmune mice are not affected by raltegravir, we consider off-target effects unlikely and attribute the exacerbation of autoimmunity to the inhibition of retroviral integrase.