- Open Access
Structural characteristics and antiviral activity of multiple peptides derived from MDV glycoproteins B and H
© Wang et al; licensee BioMed Central Ltd. 2011
- Received: 14 January 2011
- Accepted: 25 April 2011
- Published: 25 April 2011
Marek's disease virus (MDV), which is widely considered to be a natural model of virus-induced lymphoma, has the potential to cause tremendous losses in the poultry industry. To investigate the structural basis of MDV membrane fusion and to identify new viral targets for inhibition, we examined the domains of the MDV glycoproteins gH and gB.
Four peptides derived from the MDV glycoprotein gH (gHH1, gHH2, gHH3, and gHH5) and one peptide derived from gB (gBH1) could efficiently inhibit plaque formation in primary chicken embryo fibroblast cells (CEFs) with 50% inhibitory concentrations (IC50) of below 12 μM. These peptides were also significantly able to reduce lesion formation on chorioallantoic membranes (CAMs) of infected chicken embryos at a concentration of 0.5 mM in 60 μl of solution. The HR2 peptide from Newcastle disease virus (NDVHR2) exerted effects on MDV specifically at the stage of virus entry (i.e., in a cell pre-treatment assay and an embryo co-treatment assay), suggesting cross-inhibitory effects of NDV HR2 on MDV infection. None of the peptides exhibited cytotoxic effects at the concentrations tested. Structural characteristics of the five peptides were examined further.
The five MDV-derived peptides demonstrated potent antiviral activity, not only in plaque formation assays in vitro, but also in lesion formation assays in vivo. The present study examining the antiviral activity of these MDV peptides, which are useful as small-molecule antiviral inhibitors, provides information about the MDV entry mechanism.
- Marek's disease virus
- plaque formation
- chorioallantoic membrane
- structural characteristics
- antiviral inhibitor
- viral entry mechanism
The entry of enveloped viruses into host cells occurs via fusion of the viral envelope with the cellular membrane. This membrane fusion is mediated by several glycoproteins in the viral envelope that overcome strong repulsive hydration forces as well as steric and electrostatic barriers. Several of the functional motifs present in different viral fusion glycoproteins are drug development targets .
Herpesviruses are structurally complex enveloped viruses that have at least twelve glycoproteins on their surfaces. Unlike orthomyxoviruses, paramyxoviruses, filoviruses, and retroviruses, which all use a single fusion glycoprotein for membrane fusion, herpesviruses use a conserved core fusion machinery consisting of the glycoprotein gB and a gH-gL heterodimer . gB is a class III viral fusion protein, also called a fusogen, that is presumably directly involved in bringing the viral and cellular membranes together but cannot function on its own [3, 4]. The crystal structure of the gH ectodomain bound to gL shows an unusually tight complex with a unique architecture; and the formation of a gB-gH-gL complex is critical for membrane fusion . The fusion machinery of herpesviruses is more complex than that of most enveloped viruses and is somewhat reminiscent of the fusion machinery involved in cellular fusion processes [6–9]. In some herpesviruses, both gH and gB possess heptad repeat (HR) regions, and the peptides from HR regions have been shown to inhibit fusion [10–12]. Furthermore, it has been shown that the α-helix rich and hydrophobic regions of viral fusion proteins may be required for efficient induction of fusion [13–16].
Marek's disease virus (MDV) has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small animal system. MDV was long thought to be related to Epstein-Barr virus (EBV), a member of the Gammaherpesvirinae family, owing to its biological properties, particularly its slow growth in culture and its ability to induce T-cell lymphoma. Electron microscopy studies of the MDV genome provided the first evidence that this double-stranded DNA virus possesses repeat structures that are characteristic of the Alphaherpesvirinae, which was later confirmed by detailed restriction mapping and sequencing of individual genes and then entire genomes. It is now known that MDV is genetically closely related to human herpesvirus 1 (herpes simplex virus type 1, HSV-1) and human herpesvirus 3 (varicella-zoster virus, VZV) . Recent advances in MDV genetics and the sequencing of the chicken genome, aided by functional genomics have increased our understanding of lytic MDV replication and the factors and mechanisms leading to latency and tumour formation [17, 18]. MDV is found in all areas of the world and particularly virulent forms of this virus frequently cause acute explosive outbreaks, despite the availability of vaccines. The non-oncogenic MDV strains used as a vaccine prevent tumour growth but do not prevent the replication of either vaccine or virulent strains, and infectious virus particles survive at room temperature for several months . To understand the molecular mechanisms of MDV entry into host cells and to potentially identify inhibitory agents, we sought to determine the functional roles of specific regions of gH and gB proteins involved in the membrane fusion process [20, 21].
MDV gH and gB have serial HR regions showing potential antiviral activity
gHH1, gHH2, gHH3, gHH5, and gBH1 have potent antiviral activities at different steps of the viral entry process
A plaque formation experiment was conducted to identify which steps in the entry process were inhibited by gB- and gH-derived peptides at a concentration of 25 μM, the concentration which induced significant inhibition, and to compare the effect of different four methods without a strong bias. Four different assays were conducted: cells were exposed to peptides at different concentrations prior to infection (cell pre-treatment), during entry (co-treatment), after viral entry (post-treatment), or when the virus was pre-incubated with the peptide for 1 hour at 37°C before attaching to the cells (virus pre-treatment). After all treatments, the cells were incubated for 5 days at 37°C in DMEM supplemented with FCS and plaque numbers were then determined. All five peptides showed potent antiviral activity in the co-treatment experiment and inhibited infection to a minor extent in other assays. These results demonstrate that at 25 μM, gHH1 was the most effective peptide in the post-treatment assay with 100% inhibition of plaque formation. Plaque formation was completely inhibited by the gHH2 peptide at 25 μM in a virus pre-treatment assay. In addition, all five peptides tested inhibited plaque formation 60-80% in the co-treatment assay at a concentration of 25 μM. Furthermore, plaque formation was completely inhibited by gHH1 at 50 μM in the co-treatment assay and plaque formations of gHH2, gHH3, gHH5, and gBH1 were nearly 20%, 20%, 13%, and 12% at 50 μM. These results indicate that gHH1 and gHH2 should be the most effective peptides from this study for small-molecule antiviral drug design to inhibit MDV entry. Finally, the HR2 region from the fusion glycoprotein (gF) of Newcastle disease virus (NDV) (NDVHR2) at 25 μM demonstrated antiviral activity with 20% plaque formation, more effective than MDV-derived peptides, when used prior to MDV entry into cells (i.e., in the cell pre-treatment assay)." NDVHR1 and gHH6 (the negative controls) did not show significant antiviral activity, demonstrating the specificity of the antiviral effect of the MDV-derived peptides used in this study. These results are shown in Figure 2d.
None of the peptides exhibit cytotoxic effects
To confirm that these peptides did not exert toxic effects on CEF cells, cell monolayers were exposed to a range of concentrations (5, 25, 50, 100, 250, 500 μM, and 1.0 mM) of each peptide for 24 hours, and the cell viability was analysed using the lactate dehydrogenase (LDH) assay. There was no statistically significant difference between the viability of the control (untreated) cells and the cells exposed to the peptides. None of the peptides exhibited cytotoxic effects at the concentrations tested (data not shown). In addition, peptides at a 1.0 mM dosage did not exhibit any side effects on MDV-uninfected or infected embryos, including no effect on embryo activity and no apparent pathological changes.
gHH1, gHH2, gHH3, gHH5, and gBH1 have potent antiviral effects on lesion formation
Structural characteristics of gHH1, gHH2, gHH3, gHH5, and gBH1 peptides
For gHH1, gBH1, and gHH3, which share a similar α-helical secondary structure, GF chromatography and CD spectroscopy were performed under the same experimental conditions. GF chromatography of gHH1 demonstrates the formation of a homotetrameric structure with a molecular mass of about 12.1 kDa, matching the sum of four peptides. The gBH1 peptide forms a homotrimeric structure with a molecular mass of 10.7 kDa, which is approximately equal to the sum of three molecules; gHH3 adopts a monomer formation with a molecular mass of 3.8 kDa (see Figure 5). Results of CD analysis of gHH1, gHH3, and gBH1 show that all three peptides adopt a standard α-helical conformation with double minima at 208 nm and 222 nm in a PBS-buffered solution, and the tendency to form α-helices is clearer in the presence of TFE (see Figure 6).
In this study, eleven potential HR regions of MDV gH and gB were identified using GOR bio-software. These regions overlap with some α-helix-enriched regions, including gHH1, gHH3 and gBH1, and with hydrophobic regions, including gHH2 and gBH1 (data not shown). MDV glycoproteins have more HR regions than herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV), which have only two HR regions in gH or gB [11, 12]. Furthermore, five peptides (gHH1, gHH2, gHH3, gHH5, and gBH1) showed potent antiviral activity in a plaque formation assay using MDV-infected CEFs and were considered for further analysis (see Figure 2c). The plaque formation studies also demonstrated that the most active peptide, gHH1, was effective both against viral entry and after virus entry, while gHH2 was most effective in the virus pre-treatment assay (see Figure 2d). The inhibitory activity of the peptides may have occurred via the peptides associating with glycoproteins gH or gB to block the conformational changes of these glycoproteins that are crucial for fusion; it is also possible that these peptides may inhibit glycoprotein binding to receptors [13, 23].
We used CEF-associated MDV instead of cell-free MDV in cell infectivity and embryo assays due to the need for consistent treatment in terms of virus titre at different times . Ever since Woodruff and Goodpasture  first introduced the technique of cultivating fowlpox virus on the CAMs of a chicken embryo, this method has been widely used in studies of virus isolation (herpesvirus) and tissue invasion by viral transformed cells and been considered as a model system to screen drugs [26, 27]. Due to the effect of serially passaged MDV on inducing varied lesions on CAMs, we used the same passage of CEF-associated MDV to study lesion formation and reduction after peptide treatment . In addition, within a range of 50 to 80 lesions per membrane, a linear relationship exists between the number of lesions and the infecting virus dilution. Therefore, 103 pfu MDV that formed a mean number of 63 lesions was selected for use in this assay. In vivo assays may be performed by counting the number of lesions appearing on CAMs after a few days (i.e., 5-6 days) after their direct in vivo (DIO) inoculation  or 10-14 days after inoculation of yolk sacs of 9-10-day-old eggs [30, 31]. In our study, infective inoculum was inoculated into yolk sacs of 6-7-day-old chicken embryos. After 9 days of additional incubation, surviving embryos were monitored for lesion formation. Although the egg still alive until day 10-13 after infection, the lesion size on CAMs was sometimes inconsistent (e.g., there was separation into large and small lesions). As a result, we used 9 days post-infection as a constant observation time, which gave the results considerable precision. The tested peptides showed potent antiviral activity in the embryo assay, and both gHH1 and gHH2 were very effective in co-treatment and post-treatment assays at a concentration of 1.0 mM (see Figure 4). Further experiments will examine the infection of maternal antibody-free 1-day old chickens (SPF chickens) with a pathogenic strain of MDV with or without peptides to study the role of these peptides on the pathogenesis of MDV.
Much established evidence has shown that the HR2 regions of fusion glycoproteins from enveloped viruses have potent and specific antiviral activities . Our previous research demonstrated that the HR2 from NDV (i.e., avian paramyxovirus-1, APMV-1) is a specific inhibitor of NDV membrane fusion that has no cross-inhibitory activity against APMV-2 . Some reports on the HR region of bovine herpesvirus type 1 (BoHV-1) have shown infection inhibition activity, which was obtained not only with other herpesviruses but also partly with NDV . In this study, we tested a highly effective inhibitor of NDV infection for its ability to inhibit the infectivity of the unrelated MDV. The results of cell infectivity and embryo assays indicated that NDVHR2 exerted effects on MDV in the specific stage of virus entry (i.e., in cell pre-treatment and embryo co-treatment assays), suggesting a potential cross-inhibitory effect of NDV HR2 in MDV infection. In addition, NDVHR1 did not exert any effect on MDV infection, supporting the specificity of the antiviral effect of the MDV-derived peptides in this paper (see Figures 2 and 4).
We further studied the structures of the peptides used in this study. The three-dimensional (3-D) structure of HSV-2 gH shows three distinct domains: the N-terminal domain that binds gL (H1 domain), the central helical domain (H2 domain) and the C-terminal β-sandwich domain (H3 domain). Six MDV gH-derived peptides (gHH1, H2, H3, H4, H5, and H6) are within the H2 domain, which is globular and mostly helical. The H2 domain contains thirteen α-helices and three short 310 helices. In addition to the helices, this domain has a β12 strand that participates in a six-stranded β-sheet within the H1B subdomain and a short β11 strand that makes a small antiparallel β-sheet with the β4 strand of the H1B subdomain . In the current study, results of GF and CD analyses showed that MDV-gHH1 adopts a homotetrameric structure with a standard α-helical conformation, consistent with the 3-D result and this tendency to form α-helices is more obvious in the presence of TFE. The ratio of ellipticities at 222 and 208 nm can be utilized to distinguish between the monomeric and oligomeric states of helices . When the θ222/θ208 ratio is approximately 0.8, the peptide is in its monomeric state, and when this ratio exceeds 1.0, the peptide is in its oligomeric state. The CD data from MDV-gHH1 reveals that gHH1 undergoes a conformational change from the oligomeric state to a monomer/oligomer equilibrium, following which it shifts towards the monomeric state with increasing concentrations of TFE (see Figures 5 and 6). Furthermore, amino-acid alignment analysis was employed to compare the corresponding domains of MDV-gHH1 with those in other α-herpesviruses. No significant antiviral activity was found in published reports. The MDV-gHH2 has a homodimeric structure and adopts a β-sheet conformation in aqueous solution, and this β-sheet tendency is more obvious in TFE solution, as highlighted by the fact that MDV-gHH2 has a more obvious tendency to oligomerize in membrane interfaces (see Figures 5 and 6). MDV-gHH2 may be important as a binding site for glycoprotein receptors, given its potent antiviral activity, its performance in the virus pre-treatment assay, and its high propensity for interfacial hydrophobicity. The secondary structure of gHH2 is similar to that of the HSV-1 internal fusion peptide (IFP) region (a.a. 377 to 397), from which the ability to partition into membranes and aggregate within them arises . However, the domains of HSV-1 a.a. 381-420 which correspond to MDV-gHH2 did not show any significant antiviral activity . Two HSV-1 peptides, a.a. 493 to 512 and a.a. 626 to 644 of HSV-1 gH, are homologous to MDV-gHH4 and MDV-gHH6, respectively. Both peptides showed highly antiviral activity and exhibited membranotropic characteristics [23, 34]. However, MDV-gHH4 and MDV-gHH6 did not show potent antiviral activity in our study. It is worth noting that the gHH1, H2, H4, and H6 peptides of MDV gH showed different antiviral functions from the corresponding domains derived from HSV-1 gH. In fact, only 23% residue identities exist between MDV gH and HSV-2 gH, and no existing analytical tools can predict the structure of MDV gH according to the resolved 3-D structure of HSV-2 gH.
It has been established that both the HR1 (a.a. 444 to 479) and HR2 (a.a. 542 to 582) domains of HSV-1 gH show potent antiviral activity in cell infectivity assays . These domains were recently studied by Chowdary et al. using x-ray crystallography. These authors' results indicated that the pre-fusion structure of HSV-2 gH did not reveal any domains with heptad repeat (HR) characteristics . The trimeric hairpin bundle, which was suggested to be characteristic of the post-fusion form of class I and class III fusogens, is absent from the gH structure, although these two domains can form helical bundles. Given that gH appears to be able to mediate cell-cell fusion in some herpesviruses, we cannot exclude the possibility that gH has some intrinsic fusogenic properties [10–16, 34]. It is possible that the conformation of gH could change during the fusion process or viral entry to expose heptad repeats not observed in the pre-fusion structure. The results of CD analysis of MDV-gHH3 (homologous to HR1 of HSV-1 gH) in the present study showed that the peptide adopts a standard α-helical conformation and that there was no effect of polarity on the monomeric state when the peptide was transferred from polar to non-polar membrane environments, similar to the GF result in aqueous solution. MDV-gHH5 (homologous to HR2 of HSV-1 gH) revealed the formation of a homotrimeric structure in polar environments and the formation of α-helical structure in lipidic solutions (see Figures 5 and 6). More importantly, our study revealed that both MDV-gHH3 and MDV-gHH5 show potent antiviral activity, not only in plaque formation assays (in vitro) but also in embryo assays (in vivo) (see Figures 2 and 4), further supporting the idea that these peptides have fusogenic properties involved in the viral entry process.
Membrane fusion is an important step in enveloped virus entry into host cells. The present study on the antiviral activity of MDV-derived peptides that are involved in the viral entry process reveals viral entry mechanisms. These peptides may be also useful as small-molecule antiviral inhibitors. It is notable that some peptides were able to block viral infection at a post-attachment entry step, suggesting that the peptides would likely be useful as oral preventive agents or as microbicides. Further studies are needed to better define the precise mechanisms of inhibition of these peptides and the specific nature and location of their interactions with viral targets. Additional issues concerning the similarities and differences between the membrane fusion mechanisms of MDV and other α-herpesviruses should also be addressed.
Prediction and analysis of fusogenic regions
The combined use of biological software prompted us to analyse the different domains of gH (GenBank Accession No. AAL37975) and gB (GenBank Accession No. AAM97699) of MDV strain RB1B in detail for potential membrane fusion-related regions. Biological software package ExPASy-Coils http://www.ch.embnet.org/software/coils/COILS_doc.html, which has been successfully used to analyse a number of viral fusion proteins, was used to study coiled-coils (see Figure 1). We chose the ExPASy-tools program (GOR software, http://www.ch.embnet.org/, as it was designed specifically to analyse secondary structures. Hydropathy plots corresponding to the sequences of MDV gH and gB were obtained using TMpred (ExPASy, Swiss Institute of Bioinformatics, http://www.ch.embnet.org and Membrane Protein eXplorer (MpeX, Stephen White laboratory, http://blanco.biomol.uci.edu/mpex). In particular, hydropathy plots were obtained using the hydropathy index of Kyte and Doolittle and the interfacial hydrophobicity scales of Wimley and White for individual residues.
The peptides from MDV gH, identified as H1, H2, H3, H4, H5, and H6, are located at amino acid (a.a.) residues 277 to 303, 331 to 362, 396 to 429, 434 to 467, 508 to 533, and 576 to 604, respectively. The peptides from MDV gB, identified as H1, H2, H3, H4, and H5, are located at a.a. residues 340 to 367, 406 to 443, 560 to 590, 657 to 686, and 775 to 883, respectively.
Primer design and gene construction
All genes were constructed using the bridging PCR method and cloned into the GST fusion expression vector pGEX-6P-I at the BamH I-Xho I restriction sites where there is a rhinovirus 3C protease cleavage site for the fusion protein (as in the commercial PreScission™ protease cleavage site). The positive plasmids were verified by direct DNA sequencing.
Protein expression and purification
Escherichia coli strain Ros, transformed with the recombinant pGEX-6p-I plasmid, was grown at 37°C in 2 × YTA to an optical density of 0.8-1.0 (OD at 590 nm) before being induced with 1 mM IPTG for 4 hours. Bacterial cells were harvested and lysed by sonication in PBS (pH 7.3). Triton X-100 was then added to a final concentration of 1% and the lysate was incubated for 30 min at 0°C. The clarified supernatants were passed through a Glutathione-Sepharose 4B column. The GST fusion protein-bound column was washed with over 10 column volumes of PBS and eluted with 3 column volumes of reduced glutathione. The GST fusion proteins were then cleaved by GST fusion rhinovirus 3C protease at 5°C for 16 hours in a 50 mM Tris-HCl buffer, pH 7.0. The cleaved proteins were then purified by affinity filtration (with the Glutathione-Sepharose 4B column) following which the column-unbound protein was extracted and concentrated by ultrafiltration with 3K membranes (Millipore). The resultant protein was dialyzed against PBS, reduced to a proper concentration by ultrafiltration and stored at -70°C for further analysis. GST fusion proteins and cleaved proteins were analysed by 15% tricine SDS-PAGE.
Preparation of MDV stock
Primary chicken embryo fibroblasts (CEFs) were grown in DMEM supplemented with 10% foetal calf serum (FCS) and were allowed to attach overnight. CEF-associated MDV strain RB1B (from Shandong Agriculture University, has been passaged multiple times in primary CEFs) was incubated for 2 hours at 37°C. Following incubation, the virus samples on the cells were replaced with DMEM supplemented with 2% FCS and the cultures were incubated for another 5 days [24, 36]. Consistent and uniform plaques were observed and counted under an Olympus microscope and images were captured using DP Controller software. CEF-associated MDV from the same passage at 2 × 104 plaque forming units (pfu) was used in both cell infectivity and chicken egg assays in this study.
Effect of the peptides on plaque formation
All of the peptides were dissolved in DMEM without FCS and used at a range of concentrations. For the antiviral activities of peptides in the co-treatment assay, 100 pfu of MDV was incubated with the peptide at different concentrations for 2 hours at 37°C. A no-peptide sample control was also prepared and this sample was regarded as 100% plaque formation. Following incubation, the virus-peptide mixtures on the cells were replaced with DMEM supplemented with 2% FCS and the cultures were incubated for 5 days. At the end of this incubation, 50% inhibitory concentrations (IC50) values were calculated.
To assess the effects of peptides with IC50 values below 12 μM on the inhibition of MDV infectivity, four different methods [13, 23] of treating cell monolayers were used: 1) Virus pre-treatment − virus was incubated in the presence of peptides at 25 μM for 1 hour at 37°C and was then titrated onto cell monolayers; 2) Cell pre-treatment − cells were incubated with peptides for 30 minutes at 4°C. Peptides were removed, and cells were washed with PBS. Following this treatment, the cells were infected with MDV; 3) Co-treatment − the cells were incubated peptides in the presence of viral inoculum for 1 hour at 37°C; and 4) Post-treatment − cell monolayers were infected with virus for 45 minute at 37°C. The peptides were then added to the inoculum, followed by an additional 30 minute incubation at 37°C. Monolayers were incubated for 5 days at 37°C in DMEM supplemented with 2% FCS. The ratio of plaque counts to the no-peptide sample control is reported as the percentage of plaque formation (by arithmetic conversion of the mean percent plaque formation). Results are expressed as the average of triplicates ± the standard deviation and all experiments were conducted in parallel with each peptides and non-specific peptides.
LDH assay for toxicity analysis
Peptide cytotoxicity was measured using the lactate dehydrogenase (LDH) assay. This assay was performed according to the manufacturer's instructions using a commercial cytotoxicity detection kit (Roche).
Virus-yield reduction assay in chicken embryos
Briefly, 1 × 103 pfu of CEF-associated MDV, was injected into yolk sacs of 6-7-day-old embryonating specific-pathogen free (SPF) chicken eggs. After 9 days of additional incubation, surviving embryos were chilled overnight at 4°C and observed for lesion formation.
In the co-treatment protocol, a mixture of the MDV inoculum (1 × 103 pfu) with various concentrations of peptide (0.1, 0.5, 1.0 mM) in 60 μl of solution was injected into the yolk sacs of chicken eggs and incubated at 37°C for 9 days. For post-treatment assays, the yolk sacs were infected with virus for 1.5 hours at 37°C and then peptides were administered over a range of concentrations for 9 days. The chorioallantoic membranes (CAMs) at day 9 post-incubation were fixed in 10% buffered formalin. Lesions (pox) were observed and counted under an Olympus microscope, and lesion images were captured using DP Controller software. The ratio of lesion counts to the no-peptide sample control is presented as the percentage of infection (by arithmetic conversion of the mean percent lesion formation). Results are expressed as the average of triplicates ± the standard deviation and all experiments were conducted in parallel with each peptides and non-specific peptides. Five embryos were used in each experiment to generate small standard errors in the assay.
Mass spectrometry (MS) analyses
All of the purified cleaved peptides were resolved in a 20 mM Tris-HCl, pH 8.0 buffer and then analysed using the Bruker Daltonics Biflex III MALDI-TOF Mass Spectrometer to ascertain the molecular masses of the peptides.
Gel filtration (GF) analyses
The purified cleaved peptides were loaded onto the Superdex G75 column in a solution buffer of 20 mM Tris-HCl, pH 8.0. The peak molecular mass was estimated by comparison with protein standards running on the same column. The peak fractions were collected and analysed by 15% SDS-PAGE. The analytical column was calibrated using a series of individual runs of standard molecular mass proteins as markers including bovine serum albumin (68 kDa), egg white albumin (43 kDa), ribose nucleotidase (13.7 kDa), aprotinin (6.5 kDa), antimicrobial peptides (5 kDa), and vitamin B12 (1.4 kDa).
Circular dichroism (CD) spectroscopy analyses
The purified, cleaved peptides were dissolved in 10 μM PBS, pH 7.4 with 20%, 40%, or 80% 2,2,2 trifluoroethanol (TFE). The wavelength-dependence of molar ellipticity [θ] was monitored at 25°C as the average of eight scans in a spectropolarimeter (Model J-710) equipped with a thermoelectric temperature controller. The TFE solution was obtained from Fluka (Sigma-Aldrich, Milan, Italy) and was prepared using distilled water. The buffers were also filtered in a vacuum pump system using 0.2 μm pore membrane filters. TFE is widely used in conformational studies because it promotes intramolecular hydrogen bonds in spite of intermolecular interactions with water molecules. Moreover, as TFE lowers the polarity of the solution, the environmental changes explored by the peptides resemble those of the native sequences during the membrane fusion process .
This work was supported by the Foundation for the Authors of National Excellent Doctoral Dissertations of PR China (FANEDD) (2006079) and supported by the Chinese Universities Scientific Fund (2009JS10). We thank Prof. Klaus Osterrieder at Free University of Berlin for invaluable material support. We thank also Prof. Zhi-Zhong Cui at Shandong Agricultural University for generously supplying samples of MDV strain RB1B.
- Eckert DM, Kim PS: Mechanisms of viral membrane fusion and its inhibition. Ann Rev Biochem 2001, 70: 777-810. 10.1146/annurev.biochem.70.1.777View ArticlePubMedGoogle Scholar
- Heldwein EE, Krummenacher C: Entry of herpesviruses into mammalian cells. Cell Mol Life Sci 2008, 65: 1653-68. 10.1007/s00018-008-7570-zView ArticlePubMedGoogle Scholar
- Backovic M, Jardetzky TS: Class III viral membrane fusion proteins. Curr Opin Struct Biol 2009, 19: 189-96. 10.1016/j.sbi.2009.02.012PubMed CentralView ArticlePubMedGoogle Scholar
- Heldwein EE, Lou H, Bender FC, Cohen GH, Eisenberg RJ, Harrison SC: Crystal structure of glycoprotein B from herpes simplex virus 1. Science 2006, 313: 217-20. 10.1126/science.1126548View ArticlePubMedGoogle Scholar
- Chowdary TK, Cairns TM, Atanasiu D, Cohen GH, Eisenberg RJ, Heldwein EE: Crystal structure of the conserved herpesvirus fusion regulator complex gH-gL. Nat Struct Mol Biol 2010, 17: 882-8. 10.1038/nsmb.1837PubMed CentralView ArticlePubMedGoogle Scholar
- Campadelli-Fiume G, Amasio M, Avitabile E, Cerretani A, Forghieri C, Gianni T, Menotti L: The multipartite system that mediates entry of herpes simplex virus into the cell. Rev Med Virol 2007, 17: 313-26. 10.1002/rmv.546View ArticlePubMedGoogle Scholar
- Gianni T, Forghieri C, Campadelli-Fiume G: The herpesvirus glycoproteins B and H.L are sequentially recruited to the receptor-bound gD to effect membrane fusion at virus entry. Proc Natl Acad Sci USA 2007, 104: 3668-73.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirschner AN, Omerovic J, Popov B, Longnecker RL, Jadetzky TS: Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion. J Virol 2006, 80: 9444-9454. 10.1128/JVI.00572-06PubMed CentralView ArticlePubMedGoogle Scholar
- Ryckman BJ, Rainish BL, Chase MC: Characterization of the human cytomegalovirus gH/gL/UL128-31 complex that mediates entry into epithelial and endothelial cells. J Virol 2008, 82: 60-70. 10.1128/JVI.01910-07PubMed CentralView ArticlePubMedGoogle Scholar
- Galdiero S, Falanga A, Vitiello M, Browne H, Pedone C, Galdiero M: Fusogenic domains in herpes simplex virus type 1 glycoptein H. J Boil Chem 2005, 280: 28632-43. 10.1074/jbc.M505196200View ArticleGoogle Scholar
- Galdiero S, Vitiello M, D'Isanto M, Falanga A, CollinS C, Raieta K, Pedone C, Browne H, Galdiero M: Analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins H and B. J Gen Virol 2006, 87: 1085-97. 10.1099/vir.0.81794-0View ArticlePubMedGoogle Scholar
- Lopper M, Compton T: Coiled-coil domains in glycoproteins B and H are involved in human cytomegalovirus membrane fusion. J Virol 2004, 78: 8333-41. 10.1128/JVI.78.15.8333-8341.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Akkarawongsa R, Pocaro NE, Case G, Kolb AW, Brandt CR: Multiple peptides homologous to herpes simplex virus type 1 glycoprotein B inhibit viral infection. Antimicrob Agents Chemother 2009, 53: 987-996. 10.1128/AAC.00793-08PubMed CentralView ArticlePubMedGoogle Scholar
- Backovic M, Jardetzky TS, Longnecker R: Hydrophobic residues that form putative fusion loops of Epstein-Barr virus glycoprotein B are critical for fusion activity. J Virol 2007, 81: 9596-9600. 10.1128/JVI.00758-07PubMed CentralView ArticlePubMedGoogle Scholar
- Galdiero S, Vitiello M, D'Isanto M, Falanga A, Cantisan M, Browne H, Pedone C, Galdiero M: The identification and characterization of fusogenic domains in herpes virus glycoprotein B molecules. Chembiochem 2008, 9: 758-67. 10.1002/cbic.200700457View ArticlePubMedGoogle Scholar
- Gianni T, Martelli PL, Casadio R, Campadelli-Fiume G: The ectodomain of herpes simplex virus glycoprotein H contains a membrane α-helix with attributes of an internal fusion peptide, positionally conserved in the Herpesviridae family. J Virol 2005, 79: 2931-2940. 10.1128/JVI.79.5.2931-2940.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Osterrieder N, Kamil JP, Schumacher D, Tischer BK, Trapp S: Marek's disease virus: from miasma to model. Nat Rev Microbiol 2006, 4: 283-294. 10.1038/nrmicro1382View ArticlePubMedGoogle Scholar
- Jarosinski KW, Tischer BK, Trapp S, Osterrieder N: Marek's disease virus: lytic replication, oncogenesis and control. Expert Rev Vaccines 2006, 5: 761-72. 10.1586/147605188.8.131.521View ArticlePubMedGoogle Scholar
- Calnek BW, Witter RL: Neoplasic disease/Marek disease, in disease of poultry. IA: Iowa State University Press, Ames; 1991:342-385.Google Scholar
- Wu P, Reed WM, Lee LF: Glycoproteins H and L of Marek's disease virus form a hetero-oligomer essential for translocation and cell surface expression. Arch Virol 2001, 146: 983-92. 10.1007/s007050170130View ArticlePubMedGoogle Scholar
- Yoshida S, Lee LF, Yanagida N, Nazerian K: Mutational analysis of the proteolytic cleavage site of glycoprotein B (gB) of Marek's disease virus. Gene 1994, 150: 303-6. 10.1016/0378-1119(94)90442-1View ArticlePubMedGoogle Scholar
- Ding J, Cui Z, Jiang S, Li Y: Study on the structure of heteropolymer pp38/pp24 and its enhancement on the bi-directional promoter upstream of pp38 gene in Marek's disease virus. Sci China C Life Sci 2008, 51: 821-6. 10.1007/s11427-008-0099-4View ArticlePubMedGoogle Scholar
- Galdiero S, Falanga A, Vitiello M, D'Isanto M, Cantisani M, Kampanaraki A, Benedetti E, Browne H, Galdiero M: Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity. Peptides 2008, 29: 1461-71. 10.1016/j.peptides.2008.04.022View ArticlePubMedGoogle Scholar
- Lee S, Ohashi K, Sugimoto C, Onuma M: Heparin inhibits plaque formation by cell-free Marek's disease viruses in vitro. J Vet Med Sci 2001, 63: 427-32. 10.1292/jvms.63.427View ArticlePubMedGoogle Scholar
- Woodruff AM, Goodpasture EW: The susceptibility of the chorio-allantoic membrane of chick embryos to infection with the fowl-pox virus. Am J Pathol 1931, 7: 209-222.PubMed CentralPubMedGoogle Scholar
- Scher C, Haudenschild C, Klagsbrun M: The chick chorioallantoic membrane as a model system for the study of tissue invasion by viral transformed cells. Cell 1976, 8: 373-82. 10.1016/0092-8674(76)90149-5View ArticlePubMedGoogle Scholar
- Siegel BV, Batts AA: Patterns of avian viral multiplication and associated membrane histopathology in the chorioallantois of developing chick embryo. Arch Gesamte Virusforsch 1957, 7: 498-505. 10.1007/BF01241966View ArticlePubMedGoogle Scholar
- Sharma JM, Coulson BD, Young E: Effect of in vitro adaptation of Marek's disease virus on pock induction on the chorioallantoic membrane of embryonated chicken eggs. Infect Immun 1976, 13: 292-295.PubMed CentralPubMedGoogle Scholar
- Longenecker BM, Pazderka F, Stone HS, Gavora JS, Ruth RF: In ovo assay for Marek's disease virus and turkey herpesvirus. Infect Immun 1975, 11: 922-31.PubMed CentralPubMedGoogle Scholar
- Buranathai C, Rodriguez J, Grose C: Transformation of primary chick embryo fibroblasts by Marek's disease virus. Virology 1997, 239: 20-35. 10.1006/viro.1997.8854View ArticlePubMedGoogle Scholar
- Yakovleva LS, Mazurenko NP, Gunenkova NK, Vinogradov VN, Pavlovskaya AI, Chernyakhovskaya IY: Marek's disease virus (Kekava strain) replication in chickens, chick embryos and cell cultures. Acta Virol 1975, 19: 293-8.PubMedGoogle Scholar
- Wang XJ, Bai YD, Zhang GZ, Wang M, Gao GF: Structure and function study of paramyxovirus fusion protein heptad repeat peptides. Arch Biochem Biophys 2005, 436: 316-322. 10.1016/j.abb.2005.02.004View ArticlePubMedGoogle Scholar
- Okazaki K, Kida H: A synthetic peptide from a heptad repeat region of herpesvirus glycoprotein B inhibits virus replication. J Gen Virol 2004, 85: 2131-7. 10.1099/vir.0.80051-0View ArticlePubMedGoogle Scholar
- Galdiero S, Falanga A, Vitiello G, Vitiello M, Pedone C, D'Errico G, Galdiero M: Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process. Biochim Biophys Acta 2010, 1798: 579-91. 10.1016/j.bbamem.2010.01.006View ArticlePubMedGoogle Scholar
- Atanasiu D, Whitbeck JC, de Leon MP, Lou H, Hannah BP, Cohen GH, Eisenberg RJ: Bimolecular complementation defines functional regions of herpes simplex virus gB that are involved with gH/gL as a necessary step leading to cell fusion. J Virol 2010, 84: 3825-34. 10.1128/JVI.02687-09PubMed CentralView ArticlePubMedGoogle Scholar
- Qian JF, Chen PY, Cai BX: Comparison of growth characteristics between Marek's disease virus and herpesvirus of turkey in chick embryonic cell culture. Chin J Virol 1992, 3: 257-261.Google Scholar
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