Environ. Sci. Technol. 2010, 44, 5437–5443
Oxidation of Virus Proteins during UV254 and Singlet Oxygen Mediated Inactivation KRISTA RULE WIGGINTON,† LAURE MENIN,‡ JONATHAN PAZ MONTOYA,† AND T A M A R K O H N * ,† E´cole Polytechnique Fe´de´rale de Lausanne (EPFL), Laboratory of Environmental Chemistry, Institute of Environmental Engineering, Station 2, 1015 Lausanne, Switzerland, and Service de spectrometrie de masse, ISIC, BCH1529, 1015 Lausanne, Switzerland
Received February 8, 2010. Accepted May 17, 2010.
Despite the widespread use of UV254 irradiation and solar disinfection for water treatment, little is known about the photochemical pathways that lead to virus inactivation by these treatments. The goal of this study was to identify reactions that occur in virus capsid proteins upon treatment by UV254 irradiation and 1O2, an important oxidant involved in sunlightmediated disinfection. Bacteriophage MS2 was inactivated via UV254 irradiation and exposure to 1O2 in buffered water, and their capsid proteins were then analyzed with MALDI-TOF-TOF and ESI-TOF before and after digestion with protease enzymes. The results demonstrate that chemical modifications occur in the MS2 major capsid protein with both treatments. One oxidation event was detected following 1O2 treatment in an amino acid residue located on the capsid outer surface. UV254 treatment caused three chemical reactions in the capsid proteins, two of which were oxidation reactions with residues on the capsid outer surface. A site-specific cleavage also occurred with UV254 irradiation at a protein chain location on the inside face of the capsid shell. We attribute this UV254 induced protein scission, which is nearly unprecedented in the literature, to a close association between the affected residues and viral RNA, an efficient UV254 absorber. These results suggest that viral protein oxidation by UV254 and 1O2 may play a role in virus inactivation and that viral inactivation may be tracked with mass spectrometric measurements.
Introduction Solar and UV254 treatment have long been used for the disinfection of waters and wastewaters, yet only little is known about the specific damage that ultimately causes virus inactivation by these treatments. Three distinct photochemical pathways can lead to sunlight and UV254 induced viral particle damage: direct, indirect endogenous, and indirect exogenous inactivation (1). With direct inactivation, functional groups within the organism that are able to absorb UVC/B light (e.g., nucleic acids) are directly damaged during irradiation. With indirect photochemical damage, sensitizers, or compounds that absorb light, transfer energy or an electron * Corresponding author phone: +41 (0)21 693 0891; fax: +41 (0)21 693 8070; e-mail:
[email protected]. † Institute of Environmental Engineering. ‡ ISIC. 10.1021/es100435a
2010 American Chemical Society
Published on Web 06/16/2010
to dissolved oxygen. The resulting species, like the reactive oxygen species (ROS) singlet oxygen (1O2) and hydroxyl radical (OH•), can subsequently damage viral constituents. Sensitizer groups occur both within the organism (indirect endogenous) and in light-absorbing materials in the water column such as natural organic matter (NOM; indirect exogenous). For virus particles treated with UV254 in clear waters, most light absorption occurs in the viral nucleic acids although proteins also contain endogenous chromophores (2). Only the aromatic side chains of tyrosine, phenylalanine, and tryptophan absorb UV254 irradiation (ε254 ) 350, 140, and 2900 M-l cm-l, respectively) as do disulfide cystines (ε254 ) 270 M-l cm-l) (3). These residues have been shown to degrade with UV254 when in free amino acid (3-5), in short peptide (5), and in full protein form (6, 7), and UV254 irradiation lead to protein cleavage in at least one report (8). UV254 absorbing residues may also act as sensitizers that produce ROS and subsequently oxidize nonabsorbing residues (9, 10). Besides its production upon irradiation by UV254 treatment, 1O2 also plays an important role in solar disinfection. In highly colored waters, like those encountered in waste stabilization ponds (WSP) or natural surface waters, solar UVB light extends only through the top layer of the photic zone, due to rapid attenuation by natural organic matter. UVA and visible light penetrate further into the photic zone and initiate indirect photochemical reactions to form ROS. Thus in highly colored natural waters, indirect damage plays the dominant role in photochemical pathogen inactivation. Kohn and Nelson demonstrated that 1O2 is the principal oxidant in MS2 inactivation in sunlit waters containing NOM (11). Reactions rapidly occur between 1O2 and cysteine, methionine, tryptophan, tyrosine, and histidine when the amino acids are in their free form in neutral waters (k ) 8.9 × 106, 1.6 × 107, 3.0 × 107, 8 × 106, and 3.2 × 107 M-1 s-1, respectively (12)). A number of oxidized amino acid products have been identified or predicted (Supporting Information Table S1), many having mass shifts of +16 Da and +32 Da due to the addition of one or two oxygens (13-15). The elemental composition of these products can differ with the state of the amino acids (single residues, dipeptides, full proteins) thus suggesting that the protein environment plays a significant role in the oxidation mechanism (14). In depth reviews on side chain oxidation by ROS, in general, and 1O2, in particular, can be found in refs 10, 16, and (17). Previous work has shown that MS2 inactivation by UV254 and 1O2 causes damage to the viral genome but that protein damage may also contribute to inactivation (18). Chemical modifications by UV254 and ROS can cause changes in protein tertiary and quaternary structure (6, 19) and subsequently lead to increased hydrophobicity, increased acidity (19), protein unfolding (6), protein cross-linking (20), changes in light optical rotating and scattering properties, and increased susceptibility to protease cleavage (18, 20). Structural alterations caused by UV and 1O2 damage have led to a loss or increase in protein function (19). Viral protein capsids play a crucial role in both virus-host interactions and genome injection; therefore, capsid damage may render the virus noninfective. The advent of protein mass spectrometric methods have provided accurate mass measurements of entire virus particles (21, 22), individual capsid proteins (23), and peptide products from protease treatment (24, 25). Additionally, tandem mass spectrometry analyses by the same instruments reveal peptide sequences. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometries (MS) have been employed to detect and VOL. 44, NO. 14, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. MS2 Capsid Protein Sequence with Predicted Cleavage Sites (Spaces) from the Three Protease Enzymes Used in This Studya
a Underlined sequences were detected with MALDI-TOF and/or ESI-TOF. Estimated in silico digest peptide masses are provided in Supporting Information Tables S2, S3, and S4.
identify virus protein peptide modifications and identify viral mutants (24). Until now, however, no studies have used mass spectrometric techniques to identify virus capsid modifications that occur due to disinfection processes. The goal of this study was to detect specific oxidation events within viral capsid proteins upon virus disinfection with UV irradiation (254 nm) or with singlet oxygen. MS2 was employed as a surrogate for nonenveloped enteric human viruses as it has been extensively employed in water treatment studies. In addition, it is easily cultured and concentrated to high concentrations (>1014 PFU/mL) and has a relatively simple capsid structure with 180 identical copies of the capsid protein (13.7 kDa, 129 residues; Table 1) and a single copy of the assembly protein (43.9 kDa, 339 residues). The capsid proteins are arranged in a T-3 isocahedral structure and pores with 1.8 nm diameters are present at the 3-fold and 5-fold axes (26). Each capsid protein copy contains several residues susceptible to UV and 1O2 oxidation including two tryptophans, two cysteines, three tyrosines, two methionines, and four phenylalanines but no histidines and no cystines. The measurement of the capsid carbonyl content as an indicator of capsid oxidation was recently proposed as a technique to track virus infectivity due to the current lack of infectivity assays for a number of nonculturable human viruses (27). Though sensitive, such methods lack the ability to identify and distinguish between different oxidization sites, which may contribute to inactivation to varying degrees. As we report here, MALDI-MS and ESI-MS analysis can rapidly and site-specifically detect protein capsid damage from UV and 1O2. This type of method would be a significant improvement over the time-consuming culture methods that are presently employed to track virus viability in culturable viruses. To our knowledge, this is the first report on the specific sites of virus capsid oxidation during drinking water treatment processes.
Materials and Methods The chemicals, materials, and microorganisms employed in this study are described in the Supporting Information. UV254 and 1O2 Inactivation Protocols. All experiments were performed in dilution buffer (DB; 2 mL) containing 6 × 1011 PFU/mL infective MS2. Triplicate samples were run in parallel in 5 mL beakers with constant stirring. Aliquots 5438
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of UV254- and 1O2-treated samples were plated for enumeration and/or treated with proteases for mass spectral analysis immediately following inactivation treatments. For UV254 inactivation experiments, samples were placed approximately 5 cm below a 30 W germicidal UV lamp (model G30T8, 2.7 ( 0.3 mW/cm2 irradiance at 253.7 nm wavelength, Sankyo Denki, Tokyo, Japan). UV irradiance was determined by actinometry (28). Samples were exposed to UV254 irradiance between 0 and 120 s, and data were corrected for light shielding by the virus particles as described previously (11). Samples were treated with 1O2 as previously described (18). In brief, samples containing the 1O2 sensitizer Rose Bengal (RB) were exposed to light emitted from a Sun 2000 Solar Simulator (ABET Technologies, Milford, Connecticut) equipped with a 1 kW Xe lamp, an AM1.5, and a UVB cutoff filter. The UVB filter was used to avoid irradiation by light in the genome-damaging wavelength range, in order to isolate the process of inactivation by 1O2. The initial RB concentration was 2.5 mg/L, and additional RB was added every 15 min to compensate for photobleaching. The resulting 1O2 concentration remained approximately constant throughout the experiment at 2.0 × 10-11 M as measured with the probe compound furfuryl alcohol (11). Samples were exposed to simulated sunlight up to 30 min, depending on the desired inactivation level. Control samples either without the addition of Rose Bengal or in the absence of light were also analyzed. Samples were treated to 1, 2, 4, and 8 log10 of inactivation with UV254 or 1O2 and then stored at 4 °C until analysis of the full protein or treated with protease enzymes for peptide analysis. Protease Treatment. Samples were digested with protease enzymes prior to mass spectrometric analysis to identify specific residues that had been altered with UV254 or 1O2 treatment. Trypsin, chymotrypsin, and Glu-C digestions were each used as they cleave at different residues (Table 1). The protease treatment protocols and in silico digestion products are provided in the Supporting Information. Mass Spectrometry Analysis. Full protein analysis was performed by positive ion linear mode MALDI-TOF (timeof-flight) mass spectrometry. Peptide measurements were performed by ESI-TOF and MALDI-TOF, and peptide fragmentation was conducted with MALDI-TOF-TOF. All MALDI
FIGURE 1. MS2 inactivation by A) UV254 and B) 1O2. Experimental conditions: [MS2]0 ) 6 × 1011 PFU/mL in 5 mM phosphate buffer, pH 7.4; [Rose Bengal] ) 2.5 mg/L (when present); [NaN3] ) 20 mM (when present). Error bars represent 95% confidence intervals. measurements were performed with an ABI 4800 MALDITOF-TOF (Applied Biosystems, Rotkreuz, Switzerland). For peptide measurements, the instrument was operated in positive-ion reflectron mode with an average collection of 900 laser shots per spectrum collected randomly across the sample spot. A 7 mg/mL solution of R-cyano-4-hydroxycinnamic acid in 50% ACN/49.9% H2O/0.1% TFA was used as the peptide sample matrix and combined with the samples in a 10:1 matrix to sample ratio. The matrix/sample mixtures were spotted on a MALDI plate and air-dried. Peptide masses were calibrated externally with a peptide calibrant mix consisting of six peptides ranging from m/z 904 to 3658 (4700 Mass Standard Kit, Applied Biosystems). Samples were scanned from 600 to 4000 m/z. For full protein measurements, the instrument was operated with approximately 2400 shots per spectrum collected randomly across the sample spot. A 14 mg/mL solution of sinapinic acid in 50% ACN/49.9% H2O/ 0.1%TFA was used as the sample matrix at 10:1 matrix to sample ratios. Spectra were calibrated externally with the insulin, ubiquitin, and cytochrome c [M + H]+ peaks. Calibrants were deposited near the full protein samples on the MALDI plate. An m/z range of 4000 to 40000 was scanned in linear mode MALDI-TOF analysis. Peptide fragmentation on selected precursors was conducted in positive ion MS/ MS mode with a collision energy of 2 kV. Approximately 4000 laser shots were applied for each precursor. MALDI spectra were analyzed with Applied Biosystems Data Explorer Software. ESI-TOF measurements were collected on a Q-TOF Ultima mass spectrometer (Waters, Milford, MA) with a z-spray ion source. The instrument was operated in positive ionization mode with 3.5 kV capillary voltage and 50 V sample cone voltage. A scan window of m/z 300 to 2000 was collected in 1 s. Samples were concentrated and desalted with C18 ZipTips (Millipore, Billerica, MA) and then injected by infusion with a solution of 50% ACN/49.8% H2O/0.2% FA at a flow rate of 20 µL/min. A phosphoric acid solution (0.01%) was used for external calibration. Spectral processing and analyses were carried out with Waters MassLynx 4.1 software.
Results and Discussion MS2 inactivation by UV254 and 1O2 were established with culturing experiments (Figure 1). The addition of a 1O2 quencher (NaN3, 20 mM) did not significantly affect the rate of MS2 inactivation by UV254, thus it was concluded that
UV-induced inactivation was not due to the formation of 1O2 via endogenous photochemical processes. No inactivation was detected in a Rose Bengal dark control or in a solar control without Rose Bengal after 30 min. Oxidation Pattern of the Whole Coat Protein. Samples treated with UV254 and 1O2 were analyzed in MALDI-TOF linear mode to detect shifts in the whole capsid protein mass. As illustrated in Figures 2 and 3, the full protein [M + H]+ and [M + 2H]2+ peaks were clearly observable in the Time ) 0 samples at m/z 13,730 and 6885, respectively. The MS2 capsid protein has a theoretical average mass of 13,728.4 g/mol, and the measured [M + H]+ was consistently within 100 ppm of the theoretical mass. A sodium adduct peak (+23) was often observed in the untreated MS2 spectra. Following 1O2 treatment, a single oxidation peak [M + H + 16]+ was observed in the whole protein spectrum (Figure 2b). This peak became visible after two log10 (99%) inactivation with 1O2 and increased in intensity relative to the [M + H]+ peak with continued inactivation. The [M + 16]+ peak did not form when samples were treated with the solar simulator in the absence of Rose Bengal. UV254 treatment also lead to a single oxidation [M + H + 16]+ peak, and additional peaks appeared at m/z +32 and +48, indicative of doubly- and triply-oxidized capsid proteins (Figure 3b). The three protein adduct peaks increased relative to the parent peak with continued inactivation. It was therefore concluded that UV254 oxidation in the MS2 capsid proteins differs from 1O2 oxidation. The three oxidation peaks at +16, +32, and +48 were also observed when NaN3 was present in the UV254 samples, thus the oxidation events were not due to endogenous 1O2 production within the capsid proteins. Native parent peaks (m/z 13,730) were still present in the sample spectra after 8 log10 UV and 1O2 inactivation (Figures 2 and 3), thus a large fraction of the virus capsid proteins remained chemically unmodified. After two log10 of UV254 inactivation, an m/z 4884 peak was detected in the full protein MALDI spectra and increased relative to the [M + H]+ and [M + 2H]2+ peaks with continued UV treatment (Figure 3a). A second peak appeared at m/z 8848 after four log10 of UV254 inactivation. The sum of the product masses, 13732, was very close to the mass of the entire protein. It was therefore concluded that a UV254 induced scission occurred. The fragmentation pattern of the m/z 4884 cleavage product (Figure S1 and Table S5) identified the sequence as Ala1-Lys43, with an additional 376 Da on the VOL. 44, NO. 14, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. A) Positive linear mode MALDI-TOF analysis of MS2 capsid protein after 0, 2, and 8 log10 inactivation by 1O2. B) Close-up of m/z 13,730 protein [M + H]+ peak and oxidation adduct formation with 1O2 treatment.
FIGURE 3. A) Positive linear mode MALDI-TOF analysis of MS2 capsid protein after 0, 2, and 8 log10 inactivation by UV254 B) Close-up of m/z 13,730 protein [M + H]+ peak and oxidation adduct formation with UV254 treatment. Lys43 residue. This additional 376 Da likely consists of Val44Thr45-Cys46 with a 75 Da adduct on the terminal Cys46 residue. The m/z 8848 cleavage product was too large to fragment with the MALDI-TOF-TOF, but the theoretical average mass of Ser47-Tyr129 is 8935.2 Da and the theoretical average mass of Val48-Tyr129 is 8848.1 Da. We therefore propose that the 8848.1 product represents residues Val48Tyr129. The presence of the 1O2 quencher NaN3 did not affect the formation of the m/z 8848 and 4884 peaks in the UV254 treated samples. Thus the formation of 1O2 via endogenous protein sensitizers was not responsible for the protein cleavage. Identification of Specific Oxidation Sites. Trypsin, chymotrypsin, and Glu-C digest samples were analyzed with MALDI-TOF-TOF and ESI-TOF to locate specific oxidized residues that were identified in the full protein analyses. Combined, the three digests resulted in 97.4% coverage of 5440
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the major capsid protein sequence (Table 1). The MALDITOF-TOF and ESI-TOF analyses conducted in this study were semiquantitative and therefore could not track minor decreases in the protein or peptide concentrations. It was possible, however, to detect major decreases in a particular peptide peak relative to other peptide peaks and to track the formation of product peaks. The majority of peaks in the ESI-TOF trypsin and chymotrypsin digest mass spectra acquired before and after eight log10 of UV254 or 1O2 inactivation had similar relative intensities, and no overall loss of ESI-TOF signal was detected (Figures S2 and S3). Similar results were obtained with the digest MALDI spectra. The prominent trypsin and chymotrypsin digest peaks that did not undergo decreases in relative peak intensities account for 76.7% of the MS2 capsid protein sequence (Ala1-Lys43, Gln49-Arg56, Thr59-Arg83, Ala107-Tyr129). We therefore conclude that the majority of residues within the MS2 capsid
FIGURE 4. Oxidized trypsin peptide Asp114-Tyr129 resulting from UV treatment. A) Product formation measured with ESI-TOF as a function of UV254 inactivation and B) MALDI-TOF-TOF fragmentation spectra of oxidation product m/z 1575.77.
FIGURE 5. A) MALDI-TOF spectra of peptide product in Glu-C digest after 8 log10 MS2 inactivation and B) MALDI-TOF-TOF fragmentation spectrum of m/z 1776.73 product. protein remained largely unaffected by UV or 1O2 treatment, even after eight log10 of MS2 inactivation. UV254 Oxidation Products. Two oxidized peptides were detected in MALDI-TOF-TOF and ESI-TOF spectra after UV254 treatment. The trypsin m/z 1559.77 peptide (Asp114-Y129) was oxidized to form an m/z 1575.77 product that was detected in both MALDI and ESI mass spectra and identified with MALDI-TOF-TOF fragmentation (Figure 4). The relative intensity of this single oxidation (+16) adduct increased with MS2 inactivation in the ESI-TOF scans (Figure 4a) though after 8 log10 of MS2 inactivation, the intensity of the +16 peak was
