Viruses and cells.
HEp-2 cells were used in all experiments unless indicated otherwise. Cells were propagated in minimum essential medium supplemented with glutamine, amphotericin B, gentamicin, and 10% fetal bovine serum (MEM 10). The A2 strain of RSV was kindly provided by Robert Channock, and working stocks of virus were prepared as described previously (8
The unpurified peptide preparations used for the initial screens (Table ) were purchased from Research Genetics (now ResGen; Huntsville, Ala.) at a stated purity of approximately 70%. The crude preparation of peptide 78-94 was synthesized and fractionated by SynPep Corporation (Dublin, Calif.). All other peptides were synthesized by 9-fluorenylmethoxy carbonyl solid-phase chemistry (Chiron Technologies, San Diego, Calif.) by the Food and Drug Administration Facility for Biotechnology Resources (Bethesda, Md.). Peptide 80-94 is a linear peptide corresponding to amino acids 80 to 94 of RhoA: ILMCFSIDSPDSLEN. Peptide 83A is the same as peptide 80-94 except for an alteration by the substitution of an alanine residue for Cys83: ILMAFSIDSPDSLEN. The sequences of the other peptides tested are shown in Table or Fig. . In general, peptides were dissolved to 10 mg/ml in stock solutions and stored in aliquots at −70°C. The solvents varied on the basis of solubility properties, as indicated in the text or Table .
Effective antiviral concentrations of RhoA-derived peptidesa
FIG. 1. The antiviral activity of a RhoA-derived peptide preparation is highest in late-eluting aggregates and aged peptide aliquots. A crude peptide corresponding to the sequence from residues 78 to 94 of RhoA was synthesized and subjected to fractionation by (more ...) Peptide purification and analysis.
Fractionation of the crude preparation of peptide 78-94 was performed at SynPep Corporation. Crude peptide was loaded onto a C18 column and eluted with a gradient of 0 to 100% acetonitrile containing 0.075% trifluoroacetic acid over 150 min. A total of 300 fractions were collected, lyophilized, and stored at −20°C until testing. The molecular weight components of active fractions were determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The N-terminal sequence and purity of the peptide product were determined by Edman sequencing on a model 494A peptide/protein sequenator (Applied Biosystems, Foster City, Calif.) by using the software of the manufacturer. Peptides 80-94 and 83A were purified to ≥95% by reverse-phase high-pressure liquid chromatography (HPLC) at the Peptide Synthesis and Analysis Unit, National Institute of Allergy and Infectious Diseases (Rockville, Md.), and mass spectrometry analysis of purified peptides was performed by the Bio-Analytical Mass Spectrometry Laboratory, National Institute of Allergy and Infectious Diseases (Rockville, Md.).
Microplaque reduction assay.
Peptides were serially diluted fourfold in MEM 10 to give final test concentrations ranging from 0.10 to 100 μg/ml. An equal volume of MEM 10 containing RSV (calculated to give a final virus concentration of 30 to 50 PFU/well) was then added to the diluted peptides. A total of 90 μl of the virus-peptide mixture was then added in triplicate to HEp-2 cells in 96-well plates that had been seeded the previous day with 1.5 × 104 to 2.0 × 104 cells per well. At 2 days postinfection, the cells were fixed in methanol and the extent of viral replication was determined by immunohistochemical staining with a mixture of anti-F monoclonal antibodies (kindly provided by Judy Beeler through the World Health Organization Reagent Bank for RSV and PIV3, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Md.).
Viral antigen reduction assay.
To simplify the quantitation of virus replication, the microplaque assay was modified in later experiments to detect virus replication by an in situ ELISA method rather than by counting of plaques. The following adaptations to the microplaque assay were made: (i) a higher inoculum of virus (≥100 PFU/well) was used; (ii) the cells were fixed on days 2 to 3 postinfection, depending on the extent of a visible cytopathic effect (CPE); and (iii) 100 μl of 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid substrate solution (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) rather than the diaminobutyric acid substrate used in the microplaque assay was added. After the substrate was allowed to develop, the absorbance of the wells was read at 405 nm with an MRX microplate reader (Dynex Technologies, Chantilly, Va.). The results were directly comparable to those obtained by the microplaque assay. Table presents the data gathered by the microplaque assay. Figures to present the data collected by the antigen reduction assay.
FIG. 3. The antiviral potency of 80-94 is dependent on the extent of cysteine oxidation. Peptide 80-94 was purified to >95% purity by reverse-phase HPLC and was then dissolved in oxidation buffer (ammonium bicarbonate buffer [pH 8.0] containing 20% DMSO) (more ...) Calculation of IC50s.
Each peptide was tested in at least two separate experiments as described above. Data from each experiment were normalized to the level for the untreated control wells. All data for each peptide were then combined and fit to a sigmoid curve by using the “regression wizard” function of SigmaPlot 2001 (SPSS Inc., Chicago, Ill.). The 50% inhibitory concentrations (IC50s) were calculated by solving the resulting curve-fit equation for the concentration corresponding to a 50% reduction in plaque number or the level of production of viral antigen compared to the plaque numbers or antigen levels in the untreated control wells.
Cell viability assay.
For the initial testing of unpurified peptide preparations (data shown in Table ), cellular cytotoxicity was assessed by visual examination of cell monolayers for CPE. In later experiments viability assays were performed by using the Cell Proliferation Assay kit from the American Type Culture Collection (Manassas, Va.). Assays were performed by using the same format used for the microplaque reduction assay or in situ ELISA, but in the absence of virus. Peptides were serially diluted in MEM 10 and were then transferred to HEp-2 cells in 96-well plates. After coincubation for 48 to 72 h, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added and the assay was performed according to the protocol of the manufacturer.
The free sulfhydryl content of peptide stocks was assessed by using Ellman's reagent (Pierce Biotechnology, Rockford, Ill.), according to the protocol of the manufacturer, adapted to a microwell format. Cysteine standards and peptide stocks were diluted 1:50 in 200 μl of reaction buffer (0.1 M sodium phosphate [pH 8.0], 1 mM EDTA) containing freshly dissolved Ellman's reagent (1 mM) in 96-well microtiter plates (Nunc International). Samples were mixed for ~30 s and were incubated for 15 min at room temperature. The absorbance at 405 nM was then measured by using an MRX microplate reader (Dynex Technologies), and free sulfhydryl values were calculated on the basis of a standard curve with concentrations in the range of 0.078 to 10 mM. All R2 values for curve fitting were within the range of 0.9990 to 1.000.
Sedimentation equilibrium experiments.
Sedimentation equilibrium analysis was performed with a Beckman Optima XL-A/I analytical ultracentrifuge with absorbance optical scanning. The cells were loaded with 130- to 145-μl volumes of sample in the optical density range of 0.3 to 0.7 absorbance units, and the absorbance was measured at either 230 or 202 nm (depending on the absorbance spectrum of the peptide). An An-60Ti 4 cell rotor was used in order to achieve the necessary centrifugal speeds cited below. Data on the sedimentation equilibrium absorbance versus the radial position were obtained at radial increments of 0.001 cm with 10 repeats at 20°C. Rotor speeds between 52,000 and 58,000 rpm were used for each sample. For a single ideal component at sedimentation equilibrium, the total solute concentration at any radial position r
) is given by the following expression:
is the concentration at a reference position, M
is the molecular weight of the solute, νbar
is the partial specific volume of the solute, ρ is the density of the sedimentation solvent, ω is the angular velocity, rref
is the radius at a reference position, R
is the gas constant, and T
is the temperature (in Kelvin). The molar mass is readily determined by nonlinear regression analysis at a single centrifugal speed or global analysis of the data obtained with multiple speeds. Classically, equation 1
was converted to a linear form given by equation 2
The weight-average molar mass is determined from the slope of the plot of ln C
. For both equations 1
the absorbance can replace the concentration for molecular weight determinations. Both nonlinear regression fitting of the data to equation 1
and the slope determined by equation 2
were used in this study since the latter provides an easy comparison of the molecular weight of the peptide on the basis of differences in the slopes. Data analysis was performed with the software package (version 4.0) provided by Beckman-Coulter Instruments linked to Origin 4.1 (Microcal Software, Inc.). The partial specific volumes of peptides 83A and 80-94 were 0.735 and 0.728 ml/g, respectively, as determined by the public domain software program Sednterp (http://www.bbri.org/RASMB/rasmb.html
) developed by D. B. Hayes, T. Laue, and J. Philo.
Gel filtration chromatography.
All chromatography was performed with an Äkta fast-protein liquid chromatography system from Amersham Biosciences (Piscataway, N.J.). Prepacked Superdex Peptide HR 10/30 columns, which have an effective separation range of 100 to 7,000 Da and a molecular mass cutoff of approximately 20,000 Da, were used for all analyses. Peptides samples were prepared as described above and were then diluted to 1 mg/ml in phosphate-buffered saline (PBS; pH 7.4) and stored at −20°C. The column was preequilibrated with two column volumes of PBS, and 50 μl of peptide solution was then loaded and eluted with PBS at a rate of 0.5 ml/min. The peptides that eluted were detected by use of UV absorbance at 214 nm. When desired, eluted peptide was collected for further analysis in 0.25-ml fractions by using an automated fraction collector (Amersham) with microplate adaptor.
Assignment of monomer and dimer elution profiles.
To verify the elution profile of monomeric peptide 80-94, purified peptide 80-94 was reacted with a fivefold molar excess of iodoacetamide (catalog no. A3221; Sigma). After 1 h of incubation at room temperature there was no detectable free sulfhydryl by the Ellman reaction. The mixture was then diluted in PBS, and the peptide was separated from unreacted iodoacetamide by size-exclusion chromatography. The peptide component eluted in single peak at a 12.01-ml elution volume and was collected for analysis by sedimentation equilibrium centrifugation. Purified peptide dimers were isolated by oxidation of peptide 80-94 in 20% dimethyl sulfoxide (DMSO), followed by elution from the size-exclusion column. The putative dimer fraction (centered at an elution volume of 10.91 ml) was collected and analyzed by sedimentation equilibrium centrifugation.