Interaction of Natural Compounds in Licorice and Turmeric with HIV-NCp7 Zinc Finger Domain: Potential Relevance to the Mechanism of Antiviral Activity.

Nucleocapsid proteins (NCp) are zinc finger (ZF) proteins, and they play a central role in HIV virus replication, mainly by interacting with nucleic acids. Therefore, they are potential targets for anti-HIV therapy. Natural products have been shown to be able to inhibit HIV, such as turmeric and licorice, which is widely used in traditional Chinese medicine. Liquiritin (LQ), isoliquiritin (ILQ), glycyrrhizic acid (GL), glycyrrhetinic acid (GA) and curcumin (CUR), which were the major active components, were herein chosen to study their interactions with HIV-NCp7 C-terminal zinc finger, aiming to find the potential active compounds and reveal the mechanism involved. The stacking interaction between NCp7 tryptophan and natural compounds was evaluated by fluorescence. To elucidate the binding mode, mass spectrometry was used to characterize the reaction mixture between zinc finger proteins and active compounds. Subsequently, circular dichroism (CD) spectroscopy and molecular docking were used to validate and reveal the binding mode from a structural perspective. The results showed that ILQ has the strongest binding ability among the tested compounds, followed by curcumin, and the interaction between ILQ and the NCp7 zinc finger peptide was mediated by a noncovalent interaction. This study provided a scientific basis for the antiviral activity of turmeric and licorice.

Discovery of Novel Pyrimidine-Based Capsid Assembly Modulators as Potent Anti-HBV Agents.

Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.

Discovery of novel HBV capsid assembly modulators by structure-based virtual screening and bioassays.

HBV capsid assembly has been regarded as an attractive potential target for anti-HBV therapy. In this study, we discovery the Novel HBV capsid assembly modulators (CAMs) through structure-based virtual screening and bioassays. A total of 16 structurally diverse compounds were purchased and assayed, including three compounds with inhibition rate > 50% at 20 µM. Further lead optimization based on the most potent compound II-1-7 (EC50 = 5.6 ± 0.1 µM) were performed by using substructure searching strategy, resulting in compound II-2-9 with an EC50 value of 1.8 ± 0.6 µM. In bimolecular fluorescence complementation (BiFC) assay, compound II-2-9 inhibited the HBV by disrupting the HBV capsid interactions. In summary, this study provides a highly efficient way to discover novel CAMs, and 2-aryl-4-quinolyl amide derivatives could serve as the starting point for development of novel anti-HBV drugs.

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.

Utilizing Potato Virus X to Monitor RNA Movement.

Mobility assays coupled with RNA profiling have revealed the presence of hundreds of full-length non-cell-autonomous messenger RNAs that move through the whole plant via the phloem cell system. Monitoring the movement of these RNA signals can be difficult and time consuming. Here we describe a simple, virus-based system for surveying RNA movement by replacing specific sequences within the viral RNA genome of potato virus X (PVX) that are critical for movement with other sequences that facilitate movement. PVX is a RNA virus dependent on three small proteins that facilitate cell-to-cell transport and a coat protein (CP) required for long-distance spread of PVX. Deletion of the CP blocks movement, whereas replacing the CP with phloem-mobile RNA sequences reinstates mobility. Two experimental models validating this assay system are discussed. One involves the movement of the flowering locus T RNA that regulates floral induction and the second involves movement of StBEL5, a long-distance RNA signal that regulates tuber formation in potato.

Screening for Components/Compounds with Anti-Rotavirus Activity: Detection of Interaction Between Viral Spike Proteins and Glycans.

Rotaviruses are the major etiologic agents of acute gastroenteritis. Viral attachment to the cell surface is crucial to initiate infection. The VP8∗ domain, the trypsinized cleavage fragment of the outermost spike protein VP4 of rotavirus, has a galectin-like structure required for binding to the cell surface. We used the evanescent-field fluorescence-assisted assay to understand the complex mechanism underlying the virus-glycan/glycoprotein interaction. Besides, we have described virus infection assays, neutralization assay, and pretreatment assay, using cell culture. These approaches using rotavirus particles will provide novel information that has been difficult to obtain from glycan microarray using recombinant VP8∗.

Breaking Down the Barriers to a Natural Antiviral Agent: Antiviral Activity and Molecular Docking of Erythrina speciosa Extract, Fractions, and the Major Compound.

The in vitro cytotoxic activity in Vero cells and the antiviral activity of Erythrina speciosa methanol extract, fractions, and isolated vitexin were studied. The results revealed that E. speciosa leaves ethyl acetate soluble fraction of the methanol extract (ESLE) was the most active against herpes simplex virus type 1 (HSV-1). Bioactivity-guided fractionation was performed on ESLE to isolate the bioactive compounds responsible for this activity. One sub-fraction from ESLE (ESLE IV) showed the highest activity against HSV-1 and Hepatitis A HAV-H10 viruses. Vitexin isolated from ESLE VI exhibited a significant antiviral activity (EC50 =35±2.7 and 18±3.3 µg/mL against HAV-H10 and HSV-1 virus, respectively), which was notably greater than the activity of the extract and the fractions. Molecular docking studies were carried out to explore the molecular interactions of vitexin with different macromolecular targets. Analysis of the in silico data together with the in vitro studies validated the antiviral activity associated with vitexin. These outcomes indicated that vitexin is a potential candidate to be utilized commendably in lead optimization for the development of antiviral agents.

Pyrimidotriazine derivatives as selective inhibitors of HBV capsid assembly.

Chronic hepatitis B virus (HBV) infection is currently treated with nucleoside/nucleotides analogs. They are potent inhibitors of HBV DNA polymerase, which also functions as reverse transcriptase. Although nucleoside/nucleotide analogs efficiently suppress HBV replication in liver cells, they cannot eradicate HBV DNA from liver cells and cure the disease. Therefore, it is still mandatory to identify and develop effective inhibitors that target a step other than reverse transcription in the viral replication cycle. HBV capsid assembly is a critical step for viral replication and an attractive target for inhibition of HBV replication. We conducted in silico screening of compounds expected to bind to the HBV capsid dimer-dimer interaction site. The selected compounds were further examined for their anti-HBV activity in vitro. Among the test compounds, novel pyrimidotriazine derivatives were found to be selective inhibitors of HBV replication in HepG2.2.15.7 cells. Among the compounds, 2-[(2,3-dichlorophenyl)amino]-4-(4-tert-butylphenyl)-8-methyl-4H,9H-pyrimido[1,2-a][1,3,5]triazin-6-one was the most active against HBV replication. Studies on its mechanism of action revealed that the compound interfered with HBV capsid assembly determined by a cell-free capsid assembly system. Thus, the pyrimidotriazine derivatives are considered to be potential leads for novel HBV capsid assembly inhibitors.

Ursolic acid: A novel antiviral compound inhibiting rotavirus infection in vitro.

Rotavirus is one of the leading causes of severe acute gastroenteritis in children under 5 years of age, mainly affecting developing countries. Once the disease is acquired, no specific treatment is available; as such, the development of new drugs for effective antirotaviral treatment is critical. Ursolic acid is a pentacyclic triterpenoid with antiviral activity, which has been studied extensively in vitro and in vivo. To study the potential antirotaviral activity of ursolic acid, its toxic potential for viral particles (virucidal effect) and cultured cells (cytotoxicity) was analysed. No effect on virion infectivity was observed with treatments of up to 40 µM ursolic acid, while incipient cytotoxicity started to be evident with 20 µM ursolic acid. The antiviral potential of ursolic acid was evaluated in in-vitro rotavirus infections, demonstrating that 10 µM ursolic acid inhibits rotavirus replication (observed by a decrease in viral titre and the level of the main viral proteins) and affects viral particle maturation (a process associated with the endoplasmic reticulum) 15 h post infection. Interestingly, ursolic acid was also found to hamper the early stages of the viral replication cycle, as a significant reduction in the number and size of viroplasms, consistent with a decrease in VP6 and NSP2 viral proteins, was observed 4 h post infection. As such, these observations demonstrate that ursolic acid exhibits antiviral activity, suggesting that this chemical could be used as a new treatment for rotavirus.

A HTRF based competitive binding assay for screening specific inhibitors of HIV-1 capsid assembly targeting the C-Terminal domain of capsid.

Due to its multifaceted essential roles in virus replication and extreme genetic fragility, the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein is a valued therapeutic target. However, CA is as yet unexploited clinically, as there are no antiviral agents that target it currently on the market. To facilitate the identification of potential HIV-1 CA inhibitors, we established a homogeneous time-resolved fluorescence (HTRF) assay to screen for small molecules that target a biologically active and specific binding pocket in the C-terminal domain of HIV-1 CA (CA CTD). The assay, which is based on competition of small molecules for the binding of a known CA inhibitor (CAI) to the CA CTD, exhibited a signal-to-background ratio (S/B) > 10 and a Z' value > 0.9. In a pilot screen of three kinase inhibitor libraries containing 464 compounds, we identified one compound, TX-1918, as a low micromolecular inhibitor of the HIV-1 CA CTD-CAI interaction (IC50 = 3.81 µM) that also inhibited viral replication at moderate micromolar concentration (EC50 = 15.16 µM) and inhibited CA assembly in vitro. Based on the structure of TX-1918, an additional compound with an antiviral EC50 of 6.57 µM and cellular cytotoxicity CC50 of 102.55 µM was obtained from a compound similarity search. Thus, the HTRF-based assay has properties that are suitable for screening large compound libraries to identify novel anti-HIV-1 inhibitors targeting the CA CTD.

Parvovirus B19 Uncoating Occurs in the Cytoplasm without Capsid Disassembly and It Is Facilitated by Depletion of Capsid-Associated Divalent Cations.

Human parvovirus B19 (B19V) traffics to the cell nucleus where it delivers the genome for replication. The intracellular compartment where uncoating takes place, the required capsid structural rearrangements and the cellular factors involved remain unknown. We explored conditions that trigger uncoating in vitro and found that prolonged exposure of capsids to chelating agents or to buffers with chelating properties induced a structural rearrangement at 4 °C resulting in capsids with lower density. These lighter particles remained intact but were unstable and short exposure to 37 °C or to a freeze-thaw cycle was sufficient to trigger DNA externalization without capsid disassembly. The rearrangement was not observed in the absence of chelating activity or in the presence of MgCl2 or CaCl2, suggesting that depletion of capsid-associated divalent cations facilitates uncoating. The presence of assembled capsids with externalized DNA was also detected during B19V entry in UT7/Epo cells. Following endosomal escape and prior to nuclear entry, a significant proportion of the incoming capsids rearranged and externalized the viral genome without capsid disassembly. The incoming capsids with accessible genomes accumulated in the nuclear fraction, a process that was prevented when endosomal escape or dynein function was disrupted. In their uncoated conformation, capsids immunoprecipitated from cytoplasmic or from nuclear fractions supported in vitro complementary-strand synthesis at 37 °C. This study reveals an uncoating strategy of B19V based on a limited capsid rearrangement prior to nuclear entry, a process that can be mimicked in vitro by depletion of divalent cations.

Design and synthesis of aminothiazole based Hepatitis B Virus (HBV) capsid inhibitors.

The capsid assembly is an essential step for Hepatitis B Virus (HBV) life cycle and is an important target for anti-HBV drug development. In this report, we identified a hit compound with aminothiazole structure by the high throughput screening (HTS) which inhibited the interaction of HBV capsid protein within the cells. The structure hopping and SAR studies of the hit compound afforded compound 79 with potent anti-HBV replication activity and good basic drug-like properties. The working mechanism studies showed that compound 79 could bind to the similar binding site of known HBV capsid inhibitor with heteroaryldihydropyrimidine (HAP) scaffold, through similar hydrophobic interactions but with a different hydrogen bond. This compound exerted potent inhibitory effect upon HBV production, either in cell culture or in mice with no obvious acute toxicity. We propose that further development of this compound could lead to novel potent anti-HBV inhibitors that target HBV capsid assembly.

A combinatorial approach of structure-based virtual screening and molecular dynamics simulation towards the discovery of a highly selective inhibitor for VP9 coat protein of Banna virus.

Structure based virtual screening of two libraries containing 27,628 numbers of antiviral compounds was used to discover a few of the potent inhibitor molecules against Banna virus (BAV). Cross-docking studies with many common interfering proteins provided five of the highly selective inhibitor for BAV. Analyses of the leading molecules with ADME-Tox filtering tool and atomistic molecular dynamics simulation studies finally discovered a benzoxazolone derivative as one of the most promising molecules towards the highly selective inhibition of BAV. The theoretical calculations are also supported by the experimental evidences where the interactions between the hit ligand and a model peptide sequence, mimicking the VP9 protein of BAV, were studied. Overall the development of a personalized therapeutic towards the highly selective inhibition of BAV is discussed herein for the first time in literature.

Astaxanthin Prevents Human Papillomavirus L1 Protein Binding in Human Sperm Membranes.

Astaxanthin (Asta), red pigment of the carotenoid family, is known for its anti-oxidant, anti-cancer, anti-diabetic, and anti-inflammatory properties. In this study, we evaluated the effects of Asta on isolated human sperm in the presence of human papillomavirus (HPV) 16 capsid protein, L1. Sperm, purified by gradient separation, were treated with HPV16-L1 in both a dose and time-dependent manner in the absence or presence of 30 min-Asta pre-incubation. Effects of HPV16-L1 alone after Asta pre-incubation were evaluated by rafts (CTB) and Lyn dislocation, Tyr-phosphorylation (Tyr-P) of the head, percentages of acrosome-reacted cells (ARC) and endogenous reactive oxygen species (ROS) generation. Sperm membranes were also analyzed for the HPV16-L1 content. Results show that HPV16-L1 drastically reduced membrane rearrangement with percentage of sperm showing head CTB and Lyn displacement decreasing from 72% to 15.8%, and from 63.1% to 13.9%, respectively. Accordingly, both Tyr-P of the head and ARC decreased from 68.4% to 10.2%, and from 65.7% to 14.6%, respectively. Asta pre-incubation prevented this drop and restored values of the percentage of ARC up to 40.8%. No alteration was found in either the ROS generation curve or sperm motility. In conclusion, Asta is able to preserve sperm by reducing the amount of HPV16-L1 bound onto membranes.

Allexiviruses may have acquired inserted sequences between the CP and CRP genes to change the translation reinitiation strategy of CRP.

Allexiviruses are economically important garlic viruses that are involved in garlic mosaic diseases. In this study, we characterized the allexivirus cysteine-rich protein (CRP) gene located just downstream of the coat protein (CP) gene in the viral genome. We determined the nucleotide sequences of the CP and CRP genes from numerous allexivirus isolates and performed a phylogenetic analysis. According to the resulting phylogenetic tree, we found that allexiviruses were clearly divided into two major groups (group I and group II) based on the sequences of the CP and CRP genes. In addition, the allexiviruses in group II had distinct sequences just before the CRP gene, while group I isolates did not. The inserted sequence between the CP and CRP genes was partially complementary to garlic 18S rRNA. Using a potato virus X vector, we showed that the CRPs affected viral accumulation and symptom induction in Nicotiana benthamiana, suggesting that the allexivirus CRP is a pathogenicity determinant. We assume that the inserted sequences before the CRP gene may have been generated during viral evolution to alter the termination-reinitiation mechanism for coupled translation of CP and CRP.

Molecular characterization of variants of a new 'rule-breaking' tobacco rattle virus RNA2 in potatoes.

In potato tubers showing pronounced corky ringspot symptoms, two related 'rule-breaking" tobacco rattle virus (TRV) RNA2s, named Da-2 and Db-2, were identified. Their coat protein (cp) genes are preceded on the 5' side by an additional gene for a 35 kDa protein for which no relationships with previously described TRV genes or their expression products were found. With estimated 4296 and 4247 nucleotides (nts) the two RNAs are the longest TRV RNA2s described so far. The difference in size between Da-2 and Db-2 is due to a duplication of a stretch of 49 nts in the 5' untranslated region of Da-2. An alignment of TRV coat proteins (cp) revealed that up to about amino acid (aa) 176 they form two rather uniform groups. The much shorter C-terminal parts of the cps, however, differ considerably in size and composition. With 56 aa this C-terminal part is much longer in the Da and Db sequences than in all other TRV cps. It differs in 18 positions in the two strains whereas their N-terminal 184 aa differ only in two positions. - In young potato plants developing from bud-cuttings of TRV Db-infected potato tubers which had been planted in soil free of virus and nematodes a gradual degradation of Db-2 was observed. In the newly formed rootlets already five days after planting a deletion of 80 nts was observed in the putative 2b gene which in other TRV strains encodes a protein necessary for nematode transmission. Thirty three days after planting the entire 2b gene, 119 nts at the 3'end of the cp gene and a portion of the original 3'untranslated region of Db-2 had been lost in the newly formed roots, leaves and stolons. The gene for the 35kDa protein was the only one which was not affected by deletions which seems to emphasize its importance for the virus. Fifty days after planting only TRV RNA1, but no TRV RNA2 were detectable.

The Potyvirus Particle Recruits the Plant Translation Initiation Factor eIF4E by Means of the VPg covalently Linked to the Viral RNA.

The viral protein genome-linked (VPg) of potyviruses is a protein covalently linked to the 5' end of viral RNA. It interacts with eIF4E, a component of the cellular translation initiation complex. It has been suggested that the 5' RNA-linked VPg could mimic the cellular mRNA cap, promoting synthesis of viral proteins. Here, we report evidence for recruitment of the plant eIF4E by Lettuce mosaic virus (LMV, potyvirus) particles via the 5' RNA-linked VPg. Analysis of the viral population was performed by enzyme-linked immunosorbent assay-based tests, either with crude extracts of LMV-infected tissues or purified viral particles. In both cases, LMV-VPg and LMV-eIF4E subpopulations could be detected. After reaching a maximum within the first 2 weeks postinoculation, these populations decreased and very few labeled particles were found later than 3 weeks postinoculation. The central domain of VPg (CD-VPg) was found to be exposed at the surface of the particles. Using a purified recombinant lettuce eIF4E and CD-VPg-specific antibodies, we demonstrate that the plant factor binds to the VPg via its central domain. Moreover, the plant eIF4E factor could be imaged at one end of the particles purified from LMV plant extracts, by immunoredox atomic force microscopy coupled to scanning electrochemical microscopy. We discuss the biological significance of these results.

Insights in luteovirid structural biology guided by chemical cross-linking and high resolution mass spectrometry.

Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses.

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.