Surface Plasmon Resonance-Based Membrane Protein-Targeted Active Ingredients Recognition Strategy: Construction and Implementation in Ligand Screening from Herbal Medicines.

Membrane proteins (MPs) are playing important roles in several biological processes. Screening new candidate compounds targeting MPs is important for drug discovery. However, it remains challenging to characterize the interactions between MPs and small-molecule ligands in a label-free method. In this study, a surface plasmon resonance (SPR)-based membrane protein-targeted active ingredients recognition strategy was constructed. This strategy contains two major modules: affinity detection module and ligand screening module. Through the combination of these two functional modules, it is feasible to screen small molecular ligands targeting MPs from herbal medicines. First, we have constructed high/low comparative C-X-C chemokine receptor type 4 (CXCR4)-expressed lentiviral particles (LVPs) models and characterized the expression levels. Then we immobilized LVPs on CM5 chips and detected the affinity between AMD3100 and CXCR4 by using affinity detection module. The KD of AMD3100 was 32.48 ± 3.17 nM. Furthermore, the suitability and robustness of the ligand screening module were validated by using AMD3100 as a positive compound. Subsequently, this module was applied in the screening of CXCR4 small molecular ligands from herbal medicine extracts. Senkyunolide I was screened out from Chuanxiong extract. The affinity constant between senkyunolide I and CXCR4 was 2.94 ± 0.36 µM. The Boyden chamber assay revealed that senkyunolide I could inhibit cell migration process. In conclusion, an SPR-based small molecular ligand recognition strategy combined with virus-based membrane protein stabilization method was constructed. The SPR-based membrane protein-targeted active ingredients recognition strategy will be an effective tool to screen target components from complex systems acting on MPs.

Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles.

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-ß-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20-30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

Graphene-Based "Hot Plate" for the Capture and Destruction of the Herpes Simplex Virus Type 1.

The study of graphene-based antivirals is still at a nascent stage and the photothermal antiviral properties of graphene have yet to be studied. Here, we design and synthesize sulfonated magnetic nanoparticles functionalized with reduced graphene oxide (SMRGO) to capture and photothermally destroy herpes simplex virus type 1 (HSV-1). Graphene sheets were uniformly anchored with spherical magnetic nanoparticles (MNPs) of varying size between ∼5 and 25 nm. Fourier-transform infrared spectroscopy (FT-IR) confirmed the sulfonation and anchoring of MNPs on the graphene sheets. Upon irradiation of the composite with near-infrared light (NIR, 808 nm, 7 min), SMRGO (100 ppm) demonstrated superior (∼99.99%) photothermal antiviral activity. This was probably due to the capture efficiency, unique sheet-like structure, high surface area, and excellent photothermal properties of graphene. In addition, electrostatic interactions of MNPs with viral particles appear to play a vital role in the inhibition of viral infection. These results suggest that graphene composites may help to combat viral infections including, but not only, HSV-1.

Poxvirus membrane biogenesis: rupture not disruption.

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane 'de novo' with free open ends exposed in the cytosol. Within the frame of the German-wide priority programme we re-addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo-EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin-layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle.

The in situ structural characterization of the influenza A virus matrix M1 protein within a virion.

The first attempt has been made to suggest a model of influenza A virus matrix M1 protein spatial structure and molecule orientation within a virion on the basis of tritium planigraphy data and theoretical prediction results. Limited in situ proteolysis of the intact virions with bromelain and surface plasmon resonance spectroscopy study of the M1 protein interaction with lipid coated surfaces were used for independent confirmation of the proposed model.

Immunodrugs: therapeutic VLP-based vaccines for chronic diseases.

Worldwide, the prevalence of noncommunicable chronic diseases is increasing. The use of vaccines to induce autoantibodies that neutralize disease-related proteins offers a means to effectively and affordably treat such diseases. Twenty vaccines designed to induce therapeutic autoantibodies were clinically tested in the past 12 years. Immunodrugs are therapeutic vaccines comprising virus-like particles (VLPs) covalently conjugated with self-antigens that induce neutralizing autoantibody responses. Four such VLP-based vaccines have been clinically tested and one has achieved proof of principle: a reduction of blood pressure in hypertensive patients. To facilitate preliminary clinical testing, novel nonclinical study programs have been developed. Safety study designs have considered the underlying B and T cell immunology and have examined potential toxicities of vaccine components and primary and secondary pharmacodynamic action of the vaccines.

Influenza A virus M1 protein structure probed by in situ limited proteolysis with bromelain.

Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation.

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs.

Aqueous extracts from peppermint, sage and lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density.

BACKGROUND: Aqueous extracts from leaves of well known species of the Lamiaceae family were examined for their potency to inhibit infection by human immunodeficiency virus type 1 (HIV-1). RESULTS: Extracts from lemon balm (Melissa officinalis L.), peppermint (Mentha x piperita L.), and sage (Salvia officinalis L.) exhibited a high and concentration-dependent activity against the infection of HIV-1 in T-cell lines, primary macrophages, and in ex vivo tonsil histocultures with 50% inhibitory concentrations as low as 0.004%. The aqueous Lamiaceae extracts did not or only at very high concentrations interfere with cell viability. Mechanistically, extract exposure of free virions potently and rapidly inhibited infection, while exposure of surface-bound virions or target cells alone had virtually no antiviral effect. In line with this observation, a virion-fusion assay demonstrated that HIV-1 entry was drastically impaired following treatment of particles with Lamiaceae extracts, and the magnitude of this effect at the early stage of infection correlated with the inhibitory potency on HIV-1 replication. Extracts were active against virions carrying diverse envelopes (X4 and R5 HIV-1, vesicular stomatitis virus, ecotropic murine leukemia virus), but not against a non-enveloped adenovirus. Following exposure to Lamiaceae extracts, the stability of virions as well as virion-associated levels of envelope glycoprotein and processed Gag protein were unaffected, while, surprisingly, sucrose-density equilibrium gradient analyses disclosed a marked increase of virion density. CONCLUSION: Aqueous extracts from Lamiaceae can drastically and rapidly reduce the infectivity of HIV-1 virions at non-cytotoxic concentrations. An extract-induced enhancement of the virion's density prior to its surface engagement appears to be the most likely mode of action. By harbouring also a strong activity against herpes simplex virus type 2, these extracts may provide a basis for the development of novel virucidal topical microbicides.

[An approach the quantitative determination of the area of glycoprotein spikes at the surface of enveloped viruses].

The density of distribution of glycoproteins on virion surface seriously influences the virus infectivity and pathogenicity. In the present work a method of quantitative determination of the area occupied by the surface glycoprotein spikes is proposed for influenza virus (strain A/PR/8/34) based on data of tritium bombardment and dynamic light scattering (DLS). The method of DLS was used for measuring the diameter of the intact virions and the subviral particles (influenza virions lacking glycoprotein spikes after bromelain digestion). The intact virions and the subviral particles were bombarded by the hot tritium atom flux followed by the analysis of the specific radioactivity of the matrix M1 protein. It was shown that the tritium label was incorporated into the amino acid residues of a thin exposed protein layer and partially penetrated through the lipid bilayer of the viral envelope. As a result, the matrix M1 protein which is located under the lipid bilayer became labeled. The tritium label distribution among different amino acid residues was the same for the M1 protein isolated from the subviral particles and the one isolated from the intact virions. This testifies that the M1 protein spatial structure remains unchanged during proteolysis of the glycoprotein spikes. The difference between the specific radioactivity of the M1 protein isolated from the intact virions and that of the M1 protein isolated from the subviral particles allowed us to calculate the portion of the viral surface which is free of the glycoprotein spikes. If approximate the influenza virion as as here the area occupied by the surface glycoproteins could be calculated. It appeared to be equal to approximately 1.4 yen 10 nm that is about 40% of the total viral surface. This is consistent with the cryoelectron tomography data published for the influenza virus (strain A/X-31). The developed approach could be applied for other enveloped high pathogenic viruses such as HIV and Ebola.

[Internal epidemic influenza virus proteins: isolation and investigation].

The internal influenza virus proteins M1 and RNP free from surface protein impurities were isolated from subviral particles (virions free from HA and NA ectomenes). The spikeless particles had no propensity to aggregate in the solution at pH 5.0 as compared with native viruses. The subviral particles of B/Hong Kong/330/01 influenza virus, which belonged to B/Victoria/2/87-lineage, were obtained by proteolytic treatment with the enzyme bromelain under the same conditions as in cases of influenza B viruses of B/Jamagata/16/88 lineage. A chromatographic analysis of the tryptic hydrolyzates obtained for matrix (M1) proteins of A(H1N1) and A(H3N2) influenza viruses revealed differences that were greatest between the protein M1 molecules isolated from influenza viruses of different subtypes of hemagglutinine. These findings suggest there are variations in the structure of this conservative internal viral protein M1 during evolution.

The raft-promoting property of virion-associated cholesterol, but not the presence of virion-associated Brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity.

Lipid rafts are enriched in cholesterol and sphingomyelin and are isolated on the basis of insolubility in detergents, such as Brij 98 and Triton X-100. Recent work by Holm et al. has shown that rafts insoluble in Brig 98 can be found in human immunodeficiency virus type 1 (HIV-1) virus-like particles, although it is not known whether raft-like structures are present in authentic HIV-1 and it is unclear whether a virion-associated raft-like structure is required for HIV replication. Independently, it was previously reported that virion-associated cholesterol is critical for HIV-1 infectivity, although the specific requirement of virion cholesterol in HIV-1 was not examined. In the present study, we have demonstrated that infectious wild-type HIV-1 contains Brij 98 rafts but only minimal amounts of Triton X-100 rafts. To directly assess the functional requirement of virion-associated rafts and various features of cholesterol on HIV-1 replication, we replaced virion cholesterol with exogenous cholesterol analogues that have demonstrated either raft-promoting or -inhibiting capacity in model membranes. We observed that variable concentrations of exogenous analogues are required to replace a defined amount of virion-associated cholesterol, showing that structurally diverse cholesterol analogues have various affinities toward HIV-1. We found that replacement of 50% of virion cholesterol with these exogenous cholesterol analogues did not eliminate the presence of Brij 98 rafts in HIV-1. However, the infectivity levels of the lipid-modified HIV-1s directly correlate with the raft-promoting capacities of these cholesterol analogues. Our data provide the first direct assessment of virion-associated Brij 98 rafts in retroviral replication and illustrate the importance of the raft-promoting property of virion-associated cholesterol in HIV-1 replication.

Detection of the potyviral genome-linked protein VPg in virions and its phosphorylation by host kinases.

The multifunctional genome-linked protein (VPg) of Potato virus A (PVA; genus Potyvirus) was found to be phosphorylated as a part of the virus particle by a cellular kinase activity from tobacco. Immunoprecipitation, immunolabeling, and immunoelectron microscopy experiments showed that VPg is exposed at one end of the virion and it is accessible to protein-protein interactions. Substitution Ser185Leu at the C-proximal part of VPg reduces accumulation of PVA in inoculated leaves of the wild potato species Solanum commersonii and delays systemic infection, which is not observed in tobacco plants. Our data show that kinases of S. commersonii differentially recognize the VPg containing Ser or Leu at position 185, whereas both forms of VPg are similarly recognized by tobacco kinases. Taken together, our data imply that the virion-bound VPg may interact with host proteins and that phosphorylation of VPg may play a role in the VPg-mediated functions during the infection cycle of potyviruses.

Native electrophoresis and Western blot analysis (NEWeB): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues.

A combination of native electrophoresis and immunodetection (Western blot) was used for the characterization of nucleoprotein particles of the potyvirus Plum pox virus (PPV). Virus particles were electrophoresed directly from plant extracts in agarose or mixed acrylamide-agarose gels under native conditions, blotted on nitrocellulose membranes, and characterized with the aid of a coat protein-specific antibody. Using this combined methodology, called NEWeB (native electrophoresis and Western blotting), we could show that a population of particles that differ in their electrophoretic mobility can be detected in extracts of Nicotiana benthamiana, that two different strains of PPV can be distinguished in double infections of the same plant and that virus particles from leaves contain detectable levels of helper component proteinase molecules. The potential of the NEWeB method for the study of structure and function of virus particles and similar nucleoprotein complexes in single and mixed infections is discussed.

Detection of potato mop-top virus capsid readthrough protein in virus particles.

Potato mop-top furovirus (PMTV) RNA 3 encodes the 20 kDa coat protein and a larger readthrough protein of 67 kDa. The readthrough protein is expressed by suppression of the amber stop codon which terminates the coat protein gene. A 21 kDa C-terminal fragment of the readthrough protein was doned, fused to glutathione S-transferase and expressed in E. coli. An antiserum prepared against purified fusion protein was used in ELISA to detect the readthrough protein in extracts of PMTV-infected leaves. Immunogold labelling studies showed that the readthrough protein was located near one extremity of some of the virus particles.

Increase of satellite tobacco ringspot virus RNA initiated by inoculating circular RNA.

A small satellite RNA of tobacco ringspot virus (sTRSV RNA) generates circular and linear molecules of unit length and repetitive sequence, linear multimers during replication. The phosphodiester junction joining the unit satellite RNA sequences in multimeric and circular RNA resisted base-catalyzed cleavage in circles but not in linear dimers. We postulate that junctions of multimeric satellite RNA form during synthesis of the polyribonucleotide chain, whereas those of circular RNA result from a ligation reaction that introduces a group blocking the junction 2'-hydroxyl. To test the relative effectiveness of linear and circular satellite RNAs in initiating replication, we inoculated onto bean (Phaseolus vulgaris cv Black Valentine) the four possible pairs of satellite RNA molecules, one member of each pair having the wild-type sTRSV RNA sequence and the other that of the replicating mutant 51AG/212CU, with each sequence provided as the unit circular or linear form. The relative amounts of wild-type and mutant satellite RNA sequence recovered from progeny virions reflected their relative abundances in the inoculum without regard to whether the sequence was supplied as a linear or a circular molecule. These results are consistent with models for the replication of the satellite RNA in which a circular form of the satellite RNA is a template for rolling circle transcription or is otherwise a replication intermediate or is readily converted to an intermediate. We also show that a circular form of a nonaccumulating satellite RNA mutant induced an increase in a satellite RNA that is endogenous to some tobacco ringspot virus virion preparations, as demonstrated previously for the linear form.

[A comparison of the protective properties of preparations of the virion and nonvirion ("soluble") antigens of the tick-borne encephalitis virus]. / Sopostavlenie protektivnykh svoistv preparatov virionnogo i nevirionnogo ("rastvorimogo") antigenov virusa kleshchevogo éntsefalita.

A comparative assessment of the protective properties of virion (VA) and nonvirion ("soluble") (NA) antigens of tick-borne encephalitis virus prepared as inactivated samples close in their parameters to vaccine preparations was carried out. The NA in the preparations free from VA or containing only trace, nonprotective amounts of it, was shown to have significantly lower protective properties than VA and exerted no booster effect on the protective activity when added to VA preparations.