The MHV and SARS-CoV synthetic peptides were synthesized by solid-phase methodology using a semi-automated peptide synthesizer and conventional N-alpha-9-fluorenylmethyloxycarbonyl chemistry by Genemed Synthesis Inc. (San Francisco, CA). Peptides were purified by reversed-phase high performance liquid chromatography, and their purity confirmed by amino acid analysis and electrospray mass spectrometry. Peptide stock solutions were prepared in 10% dimethyl sulfoxide (DMSO, spectroscopy grade): 90% H₂O (v/v), and concentrations determined spectroscopically (SmartSpec™ 3000, BioRad, Hercules, CA).

Vero E6 and L2 cells (American Type Culture Collection, Manassas, VA) were maintained in minimum essential medium (MEM) or Dulbecco modified Eagle medium (DMEM), respectively, and supplemented with 5% fetal bovine serum (FBS), penicillin G (100 U/ml), streptomycin (100 mg/ml) and 2 mM l-glutamine, at 37 °C in 5% CO₂. SARS-CoV strain Urbani was propagated in Vero E6 cells, and MHV strain A59 (ATCC, VR764) was propagated in L2 cells.

For plaque reduction assays, Vero E6 cells or L2 cells were seeded at a density of 1 × 10⁶ cells in each well of a 6-well plate 24 h (h) prior to infection. Approximately 50 plaque forming units (PFU) of either SARS-CoV strain Urbani or MHV were pre-incubated with peptide or without peptide (diluent control) in serum-free MEM (SARS-CoV) or serum-free DMEM (MHV) for 1 h at 37 °C. Vero E6 or L2 cells were then infected with (1) peptide- or vehicle-treated SARS-CoV inoculum or (2) peptide- or vehicle-treated MHV inoculum, respectively. After 1 h adsorption, the inoculum was removed, cells were washed twice with 1× phosphate buffered saline (PBS), and then overlaid with 10% FBS/MEM or 10% FBS/DMEM, both containing 0.5% Noble Agar (Sigma, Rockland, ME). L2 cells were fixed 2 days p.i. with 3.7% formalin for 2 h at room temperature and then stained with 1× crystal violet to visualize plaques. Vero E6 cells were stained with neutral red 3 days p.i. and plaque numbers were determined 24 h later.

Cell viability was determined by using the TACS™ MTT Assay (R&D Systems Inc., Minneapolis, CA) according to the manufacturer's instructions. Briefly, Vero E6 cells or L2 cells were seeded at a density of 4 × 10⁵ cells in each well of a 96-well plate 24 h prior to treatment. Cells were subsequently treated with medium, diluent-control or 100 μg/ml (∼30 μM) of peptide for 2 h prior to the addition of the tetrazolium compound MTT. Following incubation and subsequent addition of detergent, absorbance was read at 570 nm with a spectrophotometer.

CD spectra were recorded on a Jasco J-810 spectropolarimeter (Jasco Inc., Easton, MD), using a 1 mm path length, 1 nm bandwidth, 16 s response time and a scan speed of 10 nm/min. All CD runs were performed at room temperature with 50 μM peptide dissolved in 10 mM phosphate (PO₄) buffer at pH 7.0. Three successive scans between 190 and 250 nm were collected and the CD data are expressed as the mean residue ellipticity, derived from the formula Θ  = (deg × cm²)/dmol.

Methods to derive general models of surface glycoproteins have been described previously (Gallaher et al., 1989). Domains with high interfacial hydrophobicity were identified with Membrane Protein eXplorer (MPeX; Stephen White laboratory; http://blanco.biomol.uci.edu/mpex). MPeX detection of membrane spanning sequences is based on experimentally determined hydrophobicity scales (White and Wimley, 1999, Wimley and White, 1996).

Data are presented as the mean ± standard error of the means (S.E.M.). Data from peptide-treated groups were compared to vehicle-treated groups and significant difference were determined by one-way analysis of variance (ANOVA) followed by Tukey's post hoc t-test (GraphPad Prism©, San Diego, CA).

Previous studies of potential peptide inhibitors of CoV have only examined the efficacy of peptides corresponding to the α helical regions (HR regions) of the S2 subunit (Bosch et al., 2004, Liu et al., 2004, Yuan et al., 2004, Zhu et al., 2004). Development of additional peptide fusion inhibitors, based on domains of the viral fusion protein other than the prominent α helices, has yet to be explored. We used a computational approach to identify regions of the SARS-CoV fusion protein that potentially interact with bilayer membranes (Wimley and White, 1996). The WWIHS is an experimentally-determined algorithm that can be used to estimate the propensity of an amino acid sequence to interact with the lipid membrane interface (Wimley and White, 1996). Five regions of high interfacial hydrophobicity within the SARS-CoV S2 region were identified using Membrane Protein eXplorer (MpeX) (http://blanco.biomol.uci.edu/mpex/), a computer program based on the WWIHS (Fig. 1A). WW-I and WW-II peptides correspond to the amino-terminus of the S2 region, predicted to contain the fusion peptide (Sainz et al., 2005b). WW-III and WW-IV peptides correspond to the loop domain and the WW-V peptide corresponds to the aromatic domain. The latter is a highly conserved region among the Coronaviridae family and is also present in class I viral fusion proteins of otherwise disparate RNA viruses, such as HIV-1 and EboV (Sainz et al., 2005a). The transmembrane domain of the S2 region also scored high on the WWIHS (Fig. 1A), but was not investigated as it is anchored within the viral membrane and not exposed during viral entry. Although the SARS-CoV S protein shares only 20–27% amino acid sequence similarity with the S protein of MHV (Rota et al., 2003), five analogously located sequences of high interfacial hydrophobicity were identified in the S2 subunit of MHV strain A59 (Fig. 1B) and strain BHK (data not shown).Fig. 1

(A) Interfacial hydrophobicity plot corresponding to sequences of the SARS-CoV strain Urbani S2 subunit (amino acids 758–1255). (B) Interfacial hydrophobicity plot corresponding to sequences of the MHV strain A59 S2 subunit (amino acids 780–1324). Interfacial hydrophobicity plot (mean values for a window of 19 residues) was generated using the WWIHS for individual residues (Wimley and White, 1996). The regions corresponding to areas of high interfacial hydrophobicity identified in both the SARS-CoV and MHV CoV S2 subunits are highlighted by black bars, labeled as WW-I–WW-V, and hydrophobicity scores (kcal/mol) are indicated above. Schematic diagram of the CoV S protein is depicted above each hydrophobicity plot, illustrating the respective domains. HR: heptad repeat, A: aromatic domain, TM: transmembrane domain. The arrows indicates the location of the minimum furin cleavage sites (Molloy et al., 1992) present in the S proteins of SARS-CoV (RNTR, residues 758–761) (Bergeron et al., 2005) and MHV (RRAHRSVS, residues 713–720) (Luytjes et al., 1987).

Synthetic peptides corresponding to the sequences with significant WWIHS scores were synthesized (Table 1 ) and examined for their ability to inhibit either SARS-CoV plaque formation on Vero E6 cells, at peptide concentrations of ∼30 μM (Fig. 2 ). SARS_WW-I and SARS-_WW-II inhibited viral plaque formation by 58 and 39%, respectively. SARS_WW-Va, however, did not show any inhibitory effect at this concentration. This peptide was of particular interest as it was modeled after the HIV-1 peptide inhibitor, Fuzeon™ (Kilby et al., 1998) and corresponds to the C-terminus of the C-helix and the aromatic domain. Previous work from our laboratory has shown that the aromatic domain of both the SARS-CoV and MHV S2 subunit partition into the membranes of lipid vesicles and are capable of compromising membrane integrity (Sainz et al., 2005a). We hypothesized that the inability of SARS_WW-Va to inhibit SARS-CoV entry may be due to its propensity to partition into the lipid interface (Sainz et al., 2005a). A WW-V derivative with a five amino acid truncation of the aromatic domain (SARS_WW-Vb, Table 1) was capable of inhibiting SARS-CoV plaque formation by 42% (Fig. 2A). Peptides corresponding to the loop region of the SARS-CoV fusion protein were the most effective at inhibiting SARS-CoV plaque formation. SARS_WW-III and SARS_WW-IV inhibited viral plaque formation by 90 and 83%, respectively (Fig. 2A). Shown in Fig. 2B–D is a representative photograph of SARS-CoV plaque formation in the presence of these two peptides. Consistent with the experimental results presented in Fig. 2A, SARS-CoV plaque efficiency was significantly inhibited in the presence of SARS_WW-III and SARS_WW-IV, as compared to vehicle-treated controls (Fig. 2C–D versus B).Table 1

Amino acid sequences of peptides corresponding to sequences of the S2 subunits of SARS-CoV or MHV with significant WWIHS scores

To confirm that the observed anti-viral activity of the peptides tested was not a consequence of cellular cytotoxicity, a MTT assay was performed as described in Section 2.4. Cells were treated with the tetrazolium compound MTT, which is reduced by metabolically active viable cells to purple formazan dye crystals. Absorbance (OD₅₇₀) of solubilized crystals in peptide-treated or diluent-treated groups can be compared to untreated controls. Vero E6 cells treated with ∼30 μM of SARS-CoV peptides showed no difference in absorbance as compared to untreated cells. Likewise, diminished cell viability was not observed in cells treated with diluent control (Fig. 2E). These data indicate that the anti-viral capacity of the SARS-CoV peptides is not a result of cell cytotoxicity, which could directly affect CoV plaque formation.

The potent anti-viral effect observed with the SARS-CoV loop peptides prompted us to test whether peptides analogous to regions located within the loop of the MHV fusion protein had similar anti-viral efficacy. As illustrated in Fig. 1B, the MHV fusion protein also contains five regions of high interfacial hydrophobicity as determined by the WWIHS. Similar to the SARS-CoV fusion protein, two of these regions (MHV_WW-III and MHV_WW-IV) are located within the MHV S2 loop region. Synthetic peptides corresponding to these two regions (Table 1) were synthesized and examined for their ability to inhibit MHV strain A59 plaque formation on L2 cells at peptide concentrations of ∼30 μM. MHV_WW-III and MHV_WW-IV inhibited viral plaque formation by 22 and 98%, respectively (data not shown). Unlike the synthetic SARS-CoV loop peptides tested (SARS_WW-III and SARS_WW-IV), only one of the MHV loop peptides (MHV_WW-IV) significantly inhibited MHV plaque formation on L2 cells. The potent anti-viral activity of MHV_WW-IV is illustrated in Fig. 3 . As compared to vehicle-treated controls, MHV plaque efficiency was significantly inhibited in the presence of MHV_WW-IV (Fig. 3A versus B). Moreover, similar to the MTT assay results observed with the SARS-CoV peptides, the MHV_WW-IV peptide was not cytotoxic at a concentration of 30 μM on L2 cells (data not shown).Fig. 3

Peptides analogous to the loop region of the MHV S2 subunit inhibit viral plaque formation. L2 cells were infected with ∼40 PFU of MHV-A59 following a 1 h incubation with (A) vehicle or 30 μM of (B) MHV_WW-IV. Cells were fixed and stained with crystal violet 2 days p.i., and cultures were photographed 24 h later. Arrows indicate representative MHV plaques.

Based on the results presented above, we chose to further examine the inhibitory potential of SARS_WW-III, SARS_WW-IV and MHV_WW-IV, as these peptides achieved levels of inhibition of greater than 70% at concentrations of ∼30 μM. Dose–response curves were generated for each peptide, where percent inhibition in viral plaque formation was plotted as function of peptide concentration (Fig. 4 ). The inhibition efficiency of these peptides was maintainable at low concentrations. The 50% inhibitory concentration (IC₅₀) for both SARS_WW-III and SARS_WW-IV was approximately 2 μM (Fig. 4A and B). The other SARS-CoV peptides were statistically ineffective at concentrations less than 30 μM (data not shown). Similarly, the IC₅₀ value for MHV_WW-IV was 4 μM (Fig. 4C). In addition, the SARS-CoV loop peptides were ineffective inhibitors of MHV infectivity, suggesting some degree of sequence-specific inhibition (data not shown).Fig. 4

Peptide-mediated inhibition of CoV plaque formation is dose dependent. Vero E6 cells were infected with SARS-CoV following a 1 h incubation with varying concentrations (0.125–60 μM) of (A) SARS_WW-III or (B) SARS_WW-IV. L2 cells were infected with MHV-A59 following 1 h incubation with varying concentrations (0.125–60 μM) of (C) MHV_WW-IV. Percent inhibition in plaque formation was calculated as: 100 − ((no. plaques in peptide-treated/no. plaques in vehicle-treated) × 100). Significant reductions in plaque numbers relative to vehicle-treated cultures are denoted by a single asterisk (p < 0.05, one-way ANOVA and Tukey's post hoc t-test).

HR peptide inhibitors of SARS-CoV or MHV have α-helical structure and inhibit viral entry through coiled coil interactions (Bosch et al., 2003, Bosch et al., 2004, Liu et al., 2004, Zhu et al., 2004). To assess the propensity of the loop peptides studied herein to adopt a defined secondary structure, peptides were examined by CD spectroscopy (Fig. 5 ). No distinct α-helical (minima at 208 and 222 nm) or β-sheet (minima at ∼218 nm) spectra were observed for any of the peptides in 10 mM PO₄ buffer pH 7.0. Rather, spectra for all peptides in buffer showed single minima at or near 200 nm indicative of a random coil.Fig. 5

CoV inhibitory peptides do not assume a defined secondary structure. CD spectra (mean residue ellipticity Θ) of the SARS-CoV peptides: (A) SARS_WW-III and (B) SARS_WW-IV or the MHV peptide: (C) MHV_WW-IV in 10 mM PO₄ buffer pH 7.0 at room temperature.