Katharine N Bossart2* Bruce A Mungall1* Gary Crameri1 Lin-Fa Wang1 Bryan T Eaton1 and Christopher C Broder2

1

CSIRO Livestock Industries Australian Animal Health Laboratory Geelong Victoria 3220 Australia

2

Department of Microbiology and Immunology Uniformed Services University Bethesda MD 20814 USA

author email corresponding writer email* Contributed equally

Virology Journal 2005

2:57doi:10.1186/1743-422X-2-57

The electronic translation of this article is the complete one and can be found online at: http://www.virologyj.com/content/2/1/57

2005 Bossart et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0) which permits unrestricted use distribution and reproduction in any mediam provided the original job is properly cited.Keywords: Paramyxovirus Hendra virus Nipah virus envelope glycoprotein fusion infection inhibition antiviral therapies

AbstractBackground

The recent emergence of four new members of the paramyxovirus family has heightened the awareness of and re-energized research on new and emerging diseases. In particular the high mortality and person to person transmission associated with the most recent Nipah virus outbreaks like well like the very recent re-emergence of Hendra virus has confirmed the importance of developing efficient therapeutic interventions. We have previously shown that peptides corresponding to the C-terminal heptad repeat (HR-2) of the fusion envelope glycoprotein of Hendra virus and Nipah virus were potent inhibitors of both Hendra virus and Nipah virus-mediated membrane fusion using recombinant expression systems. In the current study we have developed shorter second generation HR-2 peptides which include a capped peptide via amidation and acetylation and two poly(ethylene glycol)-linked (PEGylated) peptides one with the PEG moity at the C-terminus and the other at the N-terminus. Here we have evaluated these peptides like well as the corresponding scrambled peptide controls in Nipah virus and Hendra virus-mediated membrane fusion and against infection by live virus in vitro.

Results

Unlike their predecessors the second generation HR-2 peptides exhibited high solubility and improved synthesis yields. Importantly both Nipah virus and Hendra virus-mediated fusion as well as live virus infection were potently inhibited by both capped and PEGylated peptides with IC50 concentrations comparable to the original HR-2 peptides whereas the scrambled modified peptides had no inhibitory effect. These data also indicate that these chemical modifications did not alter the functional properties of the peptides as inhibitors.

Conclusion

Nipah virus and Hendra virus infection in vitro can be potently blocked by specific HR-2 peptides. The improved synthesis and solubility characteristics of the second generation HR-2 peptides will facilitate peptide synthesis for pre-clinical trial request in an animal model of Henipavirus infection. The applied chemical modifications are also predicted to increase the serum half-life in vivo and should increase the opportunity of success in the development of an efficient antiviral therapy.Background

Two novel zoonotic paramyxoviruses have emerged to cause disease in the past decade Hendra virus (HeV) in Australia in 19945 [1] and Nipah virus (NiV) in Malaysia in 1999 [2]. HeV and NiV caused severe respiratory and encephalitic disease in animals and humans (reviewed in [34]) HeV was transmitted to humans by close contact with infected horses; NiV was passed from infected pigs to humans. Both are unusual between the paramyxoviruses in their capacity to infect and cause potentially fatal disease in a number of host species including humans. Both viruses also have an exceptionally large genome and are genetically closely related yet definite from all other paramyxovirus family members. Due to their unique genetic and biological properties HeV and NiV have been classified as prototypic members of the new genus Henipavirus in the family Paramyxoviridae [56]. Serological surveillance and virus isolation studies indicated that HeV and NiV reside surely in flying foxes in the genus Pteropus (reviewed in [7]). Investigation of possible mechanisms precipitating their emergence indicates ecological changes resulting from deforestation human encroachment into bat habitats and high intensity livestock farming practices as the likely primary factors [7]. Because these viruses are harboured in a mammalian reservoir whose range is vast both HeV and NiV have the cap capacity to cause disease through a large area and in new regions where disease was not seen previously. There have been several other suspected NiV occurrences since its recognition in 1999. Recently two confirmed outbreaks in 2004 in Bangladesh caused fatal encephalitis in humans and for the first time person-to-person transmission appeared to have been a primary mode of spread [8-12]. In addition there appeared to be directly transmission of the virus from the flying fox to humans and the case mortality rate was ~70%; significantly higher than any other NiV outbreak to date. Currently HeV and NiV are categorized as biological safety level-4 (BSL-4) pathogens and NiV has also been classified as a category C priority pathogen. Category C agents include emerging pathogens that could be engineered for mass dissemination in the future since of availability; leisure of production and dissemination; and potential for high morbidity and mortality and major health impact. All of the a through reasons illustrate why an efficient antiviral therapy is needed for henipaviruses.

Paramyxoviruses include two membrane-anchored glycoproteins that are required for virion accessory to and fusion with the membrane of the host cell. Depending on the biological properties of the virus the accessory protein is referred to as either the hemagglutinin-neuraminidase (HN) the hemagglutinin (H) or the G glycoprotein which lacks hemagglutinating and neuraminidase activities. Whereas most paramyxoviruses employ sialic acid moieties as receptors HeV and NiV make use of a cell-surface expressed protein and their G glycoprotein binds to ephrin-B2 on host cells [13]. The fusion protein (F) facilitates the fusion of virion and host cell membranes during virus infection and is an oligomeric homotrimer [1415]. The biologically energetic F protein consists of two disulfide linked subunits F1 and F2 which are generated by the proteolytic cleavage of a precursor polypeptide known as F0 (reviewed in [1617]). In all cases the membrane-anchored subunit F1 contains a new amino terminus that is hydrophobic and highly conserved along virus families and referred to as the fusion peptide (reviewed in [18]). There have been consider capable advances in the accord of the structural features and development of mechanistic models of how several viral envelope glycoproteins function in driving the membrane fusion reaction (reviewed in [19-21]). One important characteristic of many of these fusion glycoproteins are two -helical domains referred to as heptad repeats (HR) that are involved in the formation of a trimer-of-hairpins structure [2223]. HR-1 is located proximal to the amino (N)-terminal fusion peptide and HR-2 precedes the transmembrane domain near the carboxyl (C)-terminus [2224-26]. For many viral fusion glycoproteins the N-terminal HR-1 forms an interior trimeric coiled-coil surrounded by three anti-parallel helices formed from HR-2 (reviewed in [18]). Both the HeV and NiV F glycoprotein HR domains have been shown to interact with each other and form the typical 6-helix coiled-coil bundles [2427].

Peptide sequences from either HR domain of the F glycoprotein of several paramyxoviruses including HeV and NiV have been shown to be inhibitors of fusion [2528-35]. Targeting this membrane fusion step of the viral infection process has garnered much attention principally lead by job on human immunodeficiency virus type 1 (HIV-1) (reviewed in [36]). Indeed the HIV-1 envelope derived peptide enfuvirtide (Fuzeon" formerly T-20) has been clinically successful [3738]. Enfuvirtide is a 36-amino acid peptide corresponding to a portion of the C-terminal HR-2 domain of the gp41 subunit of the envelope glycoprotein. Approved by the FDA in March 2003 enfuvirtide has been shown to be compar capable to other anti-retroviral therapeutics in terms of reducing viral load and is generally well tolerated despite its parenteral administration and enfuvirtide has added significantly to optimized combination therapy in a growing number of patients with multiple HIV-1 resistance to the currently avail capable antiretroviral drugs [39].

No therapeutic treatments are currently available for HeV or NiV infection. In our previous studies we demonstrated that peptides derived from the HR-2 of either the HeV or NiV F were potent inhibitors of fusion [34]. However although these peptides were effective their specific properties such as overall length where not optimized and they were large and somewhat insoluble making synthesis and purification problematic. In preparation to appraise these peptides as potential therapeutic fusion inhibitors against NiV and HeV infection second generation versions were designed with changes aimed at improving their solubility and in vivo half-life when administered to animals. In the current study we have produced shorter 36 amino acid capped peptides by amidation at the N-terminus and acetylation at the carboxyl-terminus. In addition two alternate peptide versions were made with the addition of a poly(ethylene glycol) moiety to either the C-terminus or the N-terminus. Here we report on the biological activity of these modified peptides and demonstrate that chemical modification increased solubility significantly without altering their biological properties of inhibiting membrane fusion. Further all three versions were capable of blocking both fusion as well as live HeV and NiV infection with IC50 concentrations in the nM range comparable to those reported with other viral systems.ResultsHeptad peptide inhibition of Hendra virus and Nipah virus-mediated cell-cell fusion

Hypothetical models of the transmembrane (F1) glycoproteins of HeV and NiV are shown in Fig. 1. The models are derived by homology modeling with the known structure of the F protein of Newcastle disease virus [40]. These models are consistent protein structures predicted by the computer algorithms PHDsec [41] and TMpred [42]. Overall the structures of the HeV and NiV F1 transmembrane subunit including the heptad repeats (HR-1 and HR-2 helices) closely resemble that of the gp41 subunit of the HIV-1 envelope glycoprotein [43-45]. The depicted circle in the background represents the F2 subunit of NiV F. Due to the structural similarities and clinical success of the gp41 heptad peptides we hypothesized that peptides derived from the HR-2 of HeV or NiV F would be effective antiviral therapies for henipavirus infection. In previous studies we evaluated the inhibition properties of 42 amino acid length peptides derived from both the N and C-terminal heptad repeats (HR-1 and HR-2) of HeV and NiV F in a vaccinia virus-based reporter gene assay that quantitatively measured cell-cell fusion mediated by the envelope glycoproteins of HeV and NiV [2534]. Although both HR-1 and HR-2 derived peptides exhibited fusion inhibitory activity the HR-2 peptide (residues 447489) was more potent and more soluble. The HeV and NiV HR-2 peptides differed at three locations (amino acids 450 479 and 480) with phenylalanine arginine and leucine in NiV replaced by tyrosine lysine and isoleucine in HeV [646]. These differences in the sequence of either peptide did not alter their homologous or heterologous inhibitory activity suggesting that either peptide possessed potential therapeutic activity to both HeV and NiV. Here we designed second generation versions of the NiV based HR-2 derived peptide with changes aimed at improving their solubility and in vivo half-life when administered to animals. Shorter 36 amino acid capped peptides were synthesized (sequence denoted as FC2 in Fig. 1) by amidation at the N-terminus and acetylation at the carboxyl-terminus modifications known to have improved in vivo half-life of Fuzeon" (Thomas Matthews Trimeris Inc. personal communication). In addition two alternate peptide versions were made with the addition of a poly(ethylene glycol) moiety to either the C-terminus or the N-terminus which improved peptide solubility during preparation and may also potentially improve the pharmacokinetics in vivo [4748].

Figure 1. Hypothetical models of the transmembrane (F1) glycoproteins of Hendra virus and Nipah virus. The models are derived by homology modeling with the known structure of the F protein of Newcastle disease virus [40]. These models are consistent protein structures predicted by the computer algorithms PHDsec [41] and TMpred [42] as described in the Methods. The heptad repeats are indicated as HR-1 (grey) and HR-2 (yellow/orange) transmembrane anchor (blue). The F2 subunit is represented by the circle behind the F1 subunit. The 36 amino acid fusion inhibitor peptide sequence used in the present study is designated as FC2 and is boxed (yellow). The equivalent location of FC2 in the HeV F1 subunit is shown for comparison.

First we examined the activity of the capped peptides on HeV and NiV-mediated membrane fusion. In previous studies un-capped heptad-derived peptides had to be dissolved initially in 100% DMSO at concentrations between 50 and 500 g/ml and then diluted in mediam in order to assert solubility. Here the capped heptad-derived peptide (capped-NiV FC2) was completely soluble and dissolved in cell culture mediam at concentrations as high as 10 mg/ml. For cell-cell fusion envelope expressing-effector cells were added to peptides prior to the addition of target cells. Shown in Fig. 2 are the dose-dependent inhibition profiles of HeV (column one) and NiV-mediated (column 2) cell-cell fusion mediated by the capped-NiV FC2 peptide in Vero (Fig. 2A) U373 (Fig. 2B) and PCI 13 (Fig. 2C) cell lines. The scrambled capped control peptide (capped-ScNiV FC2) had no inhibitory effect through the same concentration range on the cell-fusion mediated by either virus in any of the three cell lines. NiV-mediated fusion appeared to be slightly more sensitive to peptide inhibition in comparison to the cell-fusion activity of HeV although the calculated IC50 concentrations in each were comparable (Table 1). Importantly the IC50 values of the capped translation of NiV FC2 in these in vitro cell-cell fusion assays were within the 1327 nM range comparable to what was observed in prior studies utilizing un-capped versions of the 42 amino acid heptad-derived peptides which yielded IC50 values between 5.2 and 5.8 nM [34].

Figure 2. Inhibition of Hendra virus and Nipah virus-mediated cell-cell fusion by capped C-terminal heptad peptide NiV FC2. HeLa cells were infected with vaccinia recombinants encoding HeV F and HeV G or NiV F and NiV G glycoproteins along with a vaccinia recombinant encoding T7 RNA polymerase (effector cells). Each designated target cell type was infected with the E. coli LacZ-encoding reporter vaccinia virus vCB21R. Each target cell type (1 105) was plated in reproduction wells of a 96-well plate. Inhibition was carried out using either capped NiV FC2 or ScNiV FC2 (control) heptad peptide. Peptides were added to the HeV or NiV glycoprotein-expressing cells (1 105) incubated for 30 min at 37 C and then each target cell type was added. The cell fusion assay was performed for 2.5 hr at 37 C followed by lysis in Nonidet P-40 (1%) and -Gal activity was quantified.

Table 1. Summary of 50% inhibitory concentration values of peptide fusion inhibitors in cell-cell fusion and virus infection assays.

Using the cell-cell fusion assay we next examined the PEG-modified versions of NiV FC2. As predicted these pegylated heptad peptides also possessed increased solubility characteristics and could be readily prepared at concentrations up to 10 mg/ml. The dose-response inhibition results of the N-PEG-NiV FC2 and C-PEG-NiV FC2 peptides are shown in Fig. 3 and inhibition was demonstrated in Vero (Fig. 3A) U373 (Fig. 3B) and PCI 13 (Fig. 3C) cell lines. Both pegylated versions of NiV FC2 were capable of blocking NiV and HeV-mediated cell-fusion while the scrambled PEG-control peptide (C-PEG-ScNiV FC2) had no inhibitory activity. Because of the required specificity of the heptad peptide amino acid sequence to convey fusion inhibitory activity as well as the high cost of peptide synthesis we chose to only synthesize one translation of the scrambled peptide as a pegylated control with the PEG10 moiety linked to the C-terminus. It was also noted that the NiV FC2 peptide with the PEG10 moiety added to the C-terminus had significantly reduced inhibitory capacity as compared to PEG10 added to the N-terminus against both NiV and HeV-mediated cell-fusion in all three cell lines tested. The reduction of C-PEG-NiV FC2 activity versus N-PEG-NiV FC2 was20-fold in all cases (Table 1) with the exception of HeV-mediated cell-fusion with the U373 cell line (Fig. 3B). Importantly in all cases the N-PEG-NiV FC2 demonstrated very similar IC50s (310 nM) to what was observed in prior studies utilizing un-capped versions of the 42 amino acid heptad-derived peptides (56 nM).

Figure 3. Inhibition of Hendra virus and Nipah virus-mediated cell-cell fusion by N-terminal and C-terminal (PEG10) pegylated heptad peptide NiV FC2. HeLa cells were infected with vaccinia recombinants encoding HeV F and HeV G or NiV F and NiV G glycoproteins along with a vaccinia recombinant encoding T7 RNA polymerase (effector cells). Each designated target cell type was infected with the E. coli LacZ-encoding reporter vaccinia virus vCB21R. Each target cell type (1 105) was plated in reproduction wells of a 96-well plate. Inhibition was carried out using either the N-terminal (N-PEG-NiV FC2) or C-terminal (C-PEG-NiV FC2) pegylated and capped heptad peptides or C-terminal pegylated scrambled control peptide (C-PEG-ScNiV FC2). Peptides were added to the HeV or NiV glycoprotein-expressing cells (1 105) incubated for 30 min at 37 C and then each target cell type was added The cell fusion assay was performed for 2.5 hr at 37 C followed by lysis in Nonidet P-40 (1%) and -Gal activity was quantified.

Heptad peptide inhibition of Hendra virus and Nipah virus infection

We next sought to verify the inhibitory activity of Nipah virus heptad-derived peptides on the infection of live HeV and NiV in cell culture. We routinely employ Vero cell culture to perform live henipavirus infection assays as well as in the propagation of virus stocks. The infection of Vero cells with HeV or NiV produced characteristic syncytial morphologies for each virus [49]. HeV reproducibly incorporated surrounding cells in the culture monolayer into each syncytium with the cell nuclei and viral proteins spread throughout the majority of the giant cell. In contrast NiV infected syncytia initially demonstrated a similar appearance to their HeV counterparts except characteristically both cell nuclei and viral protein were later sequestered round the periphery of each giant cell leaving the central region largely empty. In order to assess the extent of viral infection we have developed an assay that will detect viral protein by immunofluorescence staining and localization of the P protein using a cross-reactive anti-P peptide-specific antiserum. Using this syncytia-based immunofluorescence infection assay we initially tested the N-PEG NiV FC2 peptide for its capacity to bar virus infection and results are shown in Fig. 4. In the absence of peptide the different syncytial morphologies of HeV and NiV- infected cells were clearly evident. In the HeV-infected syncytia (Fig. 4A) the viral P protein was spread throughout the majority of the giant cell; whereas the NiV-infected syncytia (Fig. 4D) were circular structures delineated by a ring of the viral antigen. Incubation of 500 nM N-PEG-NiV FC2 with either HeV (Fig. 4B) or NiV (Fig. 4E) infected cells resulted in a dramatic and robust reduction in syncytial size although the number of syncytia per cell monolayer remained largely unchanged. In parallel the incubation of 500 nM C-PEG-ScNiV FC2 control peptide with HeV or NiV-infected cells (Fig. 4C and 4F respectively) revealed a syncytial morphology and size identical to those observed in the absence of any peptide.

Figure 4. Immunofluorescence-based syncytia assay of Hendra virus and Nipah virus infection. Vero cells were plated into 96 well plates and grown to 90% confluence. Cells were pre-treated with heptad peptides for 30 min at 37 C prior to infection with 1.5 103 TCID50/ml and 7.5 102 TCID50/ml of live HeV or NiV (combined with peptide). Cells were incubated for 24 hours fixed in methanol and immunofluorescently stained for P protein prior to digital microscopy. Images were obtained using an Olympus IX71 inverted microscope coupled to an Olympus DP70 high resolution color camera and all images were obtained at an original magnification of 85 . Representative images of FITC immunofluorescence of anti-P labeled HeV and NiV syncytia are shown. A: HeV without peptide. B: HeV with C-PEG-NiV FC2. C: HeV with N-PEG-ScNiV FC2. D: NiV without peptide. E: NiV with N-PEG-NiV FC2. F: NiV with N-PEG-ScNiV FC2.

Figure 5. Inhibition of Hendra virus and Nipah virus infection by capped heptad peptides. Vero cells or PCI 13 cells were plated into 96 well plates and grown to 90% confluence. Cells were pre-treated with the indicated peptide for 30 min at 37 C prior to infection with 1.5 103 TCID50/ml and 7.5 102 TCID50/ml of live HeV or NiV (combined with peptide). Cells were incubated for 24 hours fixed in methanol and immunofluorescently labeled for P protein prior to digital microscopy and image analysis to determine the relative area of each syncytium (see Methods). The figure shows the relative syncytial area (pixel2) versus the indicated peptide concentration for HeV and NiV.

Figure 6. Inhibition of Hendra virus and Nipah virus infection by N-terminal and C-terminal pegylated heptad peptides. Vero cells or PCI 13 cells were plated into 96 well plates and grown to 90% confluence. Cells were pre-treated with the indicated peptide for 30 min at 37 C prior to infection with 1.5 103 TCID50/ml and 7.5 102 TCID50/ml of live HeV or NiV (combined with peptide). Cells were incubated for 24 hours fixed in methanol and immunofluorescently labeled for P protein prior to digital microscopy and image analysis to determine the relative area of each syncytium (see Methods). The figure shows the relative syncytial area (pixel2) versus the indicated peptide concentration for HeV and NiV.