Reactivity of Enveloped Virus Genome, Proteins, and Lipids with Free

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Reactivity of Enveloped Virus Genome, Proteins, and Lipids with Free Chlorine and UV254 Yinyin Ye, Pin Hsuan Chang, John Hartert, and Krista R. Wigginton Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00824 • Publication Date (Web): 09 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Environmental Science & Technology

Reactivity of Enveloped Virus Genome, Proteins, and Lipids with Free Chlorine and UV254

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Yinyin Ye,† Pin Hsuan Chang,† John Hartert,† Krista R. Wigginton*,†

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†Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI

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48109-2125, United States

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*Corresponding author

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Mailing address: Department of Civil and Environmental Engineering, 1351 Beal Ave., 181

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EWRE, Ann Arbor, MI 48109-2125, USA. Phone: +1 (734) 763-2125; Fax: +1 (734) 764-4292;

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E-mail: [email protected]

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Word Counts

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5490 words in Text + 1500 words in Figures + 0 words in Table + 83 words in Acknowledgements

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= Total 7073

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ACS Paragon Plus Environment

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ABSTRACT

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The survivability of viruses in natural and engineered systems impacts public health. Inactivation

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mechanisms in the environment have been described for nonenveloped viruses, but it remains

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unclear how the membrane layer of enveloped viruses influences inactivation. We applied

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molecular tools and high-resolution mass spectrometry to measure reactions in the genome,

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proteins, and lipids of enveloped Pseudomonas phage Phi6 during inactivation by free chlorine

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and UV254. Free chlorine readily penetrated the lipid membrane to react with proteins in the

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nucleocapsid and polymerase complex. The most reactive Phi6 peptides were approximately 150

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times more reactive with free chlorine than the most reactive peptides reported in nonenveloped

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coliphage MS2. The inactivation kinetics of Phi6 by UV254 was comparable with those of

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nonenveloped adenovirus and coliphage MS2, and were driven by UV254 reactions with viral

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genomes. Our research identifies molecular features of an enveloped virus that are susceptible to

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chemical oxidants or UV radiation. Finally, the framework developed in the manuscript for

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studying molecular reactivities of Phi6 can be adopted to investigate enveloped virus survivability

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under various environmental conditions.

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INTRODUCTION

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Viruses that are transmitted through direct person-to-person contact or in large respiratory

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droplets do not typically survive for very long outside of their host in the environment.1 On the

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other hand, viruses that are transmitted through aerosols or by contact with water, food, and solid

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surfaces tend to survive longer in order to come into contact with their next host.2,3 Survivability,

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often reported as the time necessary for 90% of a population to lose infectivity (T90), can therefore

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vary widely amongst different viruses, and depends on environmental conditions including

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temperature,4-6 relative humidity,4-9 UV radiation,10-12 and oxidants.13-15 Survivability is also

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impacted by virus structures. Nonenveloped viruses are generally considered more stable than

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enveloped viruses in the environment. For example, the T90 values for nonenveloped viruses in

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wastewater range from days to months, whereas the T90 values for enveloped viruses range from

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several hours to days.16-18 In terms of susceptibility to chemical disinfectants, the enveloped Ebola

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and Phi6 viruses experience higher levels of inactivation than the nonenveloped MS2 and M13

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viruses when exposed to 0.5% NaOCl.19 Even closely related enveloped viruses can have varied

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survivabilities. For example, the T90 of severe acute respiratory syndrome (SARS) coronavirus in

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serum-free culture media is 9 days, whereas the T90 of human coronavirus 229E is less than 1 day

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under the same conditions.20 The mechanistic reasons for the higher susceptibility of enveloped

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viruses to inactivation in aqueous environments are mostly unknown.

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Despite their greater susceptibility to environmental conditions, many enveloped viruses do

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undergo environmental transmission. SARS coronaviruses were transmitted through airflow and

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virus-laden fecal aerosols.21,22 Human influenza viruses retain their infectivity on the nonporous

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surfaces, increasing their chances to infect.23,24 Avian influenza viruses are shed into water, where

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they can survive months before being consumed by their next host.25,26 Porcine reproductive and


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respiratory syndrome viruses can travel several kilometers via aerosols, spreading swine diseases

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from farm to farm.27 Infectious Ebola viruses persist in patient blood, feces and urine, and can

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survive in liquid for several days.3,28

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Research on what makes enveloped viruses more or less persistent in the environment and

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through disinfection processes could help with predicting risks posed by newly emerging viruses

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that are difficult or dangerous to culture. For example, in the recent Ebola outbreak, a mechanistic

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understanding of enveloped virus inactivation would have helped scientists predict how long Ebola

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virus remained infective in wastewater, blood, and vomit.29 Comprehensive inactivation

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mechanisms have been published for a limited number of nonenveloped viruses with

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disinfectants,12,15,30,31 but are lacking for enveloped viruses. In general, UV radiation targets

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nonenveloped virus genomes, whereas chemical oxidants inactivate nonenveloped viruses by

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genome or protein reactions, depending on the virus and oxidant.

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Recent fate and survival studies of enveloped viruses in the environment have adopted

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Pseudomonas virus Phi6 as a model enveloped virus.17,32-36 Phi6 is a double stranded RNA virus.

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The Phi6 particle is 85 nm in diameter37 and contains 11 different viral proteins.38 Like influenza

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viruses, Phi6 is enveloped, has a segmented genome, and contains glycerophospholipids in its

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envelope.39,40 In addition, it is the best studied virus in the family Cystoviridae, which includes the

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only bacteriophages that have a lipid outer layer. Moreover, Phi6 is easier to work with than other

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enveloped viruses and can be propagated to high titer levels.

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To develop a better mechanistic understanding of enveloped virus inactivation, we employed

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Phi6 in an initial investigation of enveloped virus reactivity with chemical oxidants and UV

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radiation. We characterized the biomolecule reactions in Phi6 following exposure to free chlorine

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and UV254. Phi6 inactivation, genome reactions, and protein and lipid reactions were quantified


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with plaque assays, real-time reverse transcription polymerase chain reactions (RT-qPCR), and

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liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. We compared the

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reaction rate constants of the Phi6 genome and proteins to those of nonenveloped viruses reported

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in earlier studies and measured under similar experimental conditions to elucidate the molecular

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features that may impact enveloped virus persistence in aqueous environments.

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MATERIALS AND METHODS

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Virus propagation and purification. Phi6 and its bacterial host Pseudomonas syringae pv.

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phaseolicola were kindly provided by Dr. Linsey Marr’s lab at Virginia Tech. To propagate Phi6,

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P. syringae was grown in Luria-Bertani (LB) medium containing 5 g L-1 NaCl at 26 °C and 180

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rpm to an optical density of 0.10 at 640 nm (i.e., when the cell density was approximately 1.8 ×

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108 cells mL-1). At that point, Phi6 was added to the bacteria at a multiplicity of infection equal to

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2 (i.e., ratio of Phi6 plaque-forming units (PFU) to P. syringae colony forming units (CFU)), and

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then incubated under the same conditions for 7 to 9 hours. Cells and debris were removed from

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the virus suspension by filtering it through 0.22 μm polyethersulfone (PES, Millipore) membranes.

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The filtered virus suspensions (~1 L) were concentrated to approximately 20 mL (i.e., ~50´

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concentration) in a lab-scale tangential flow filtration system (Millipore) outfitted with a 30 kDa

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cellulous filter. The concentrate was purified in a 10-40% (w/v) step sucrose gradient (average

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65,700 × g, 1.5 h, 4 °C), then in a 40-60% (w/v) linear sucrose gradient (average 65,700 × g, 15 h,

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4 °C). The phage band was collected with a needle and the buffer was exchanged for 5 mM

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phosphate buffer (PBS; 10 mM NaCl, pH 7.4) with a 100 kDa Amicon Ultra-15 centrifugal filter

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(Millipore). Virus purity was confirmed by SDS-PAGE with 8-16% TGXTM precast protein gels

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(Bio-Rad), according to the manufacturer’s instructions (Figure S1). The final Phi6 stocks (1012


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PFU mL-1) were filter-sterilized with 0.22 μm PES membranes, aliquoted, and stored at -80 °C

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until use.

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Free chlorine and UV254 experiments. Experimental virus solutions were prepared by diluting

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Phi6 in PBS. All free chlorine and UV254 experiments were conducted at room temperature.

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Infectious virus concentrations (PFU mL-1) were measured immediately before and after the

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viruses were exposed to chlorine and UV254 via plaque assays on LB agar plates.41 Samples were

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stored on ice during the plaque assays. Following free chlorine and UV treatment, samples were

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immediately stored at -80 °C prior to nucleic acid, protein, and lipid analyses.

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Free chlorine experiments. Free chlorine was prepared by diluting NaClO stock solution

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(Sigma-Aldrich) in PBS. At pH 7.4, HOCl and OCl— are equal in molar concentrations. Free

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chlorine disinfection was conducted in a modified continuous quench-flow system that was

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described previously for ozone reactions (Figure S2).42 In brief, free chlorine and Phi6 solutions

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were continuously mixed in a PEEK micro static mixing tee (IDEX Health & Science) at flow

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rates of 0.125 mL min-1 each to reach initial reaction conditions of 2 mg L-1 free chlorine as Cl2

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and 4-5 × 1010 PFU mL-1 Phi6. The reacting mixture then passed through sample loops with varied

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volumes to reach contact times of 0.3, 0.6, 2, 4, 8 and 11 s. The reactions were quenched with 550

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mM Tris-HCl (pH 7.4) at a flow rate of 0.025 mL min-1. Control experiments demonstrated that

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the addition of Tris-HCl as a quenching agent for free chlorine effectively halted Phi6 inactivation

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(Figure S3). Approximately 2.4 mL of the quenched samples were collected for nucleic acid,

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protein, and lipid analyses. After each experiment, the quench-flow system was thoroughly rinsed

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with chlorine-demand-free water. Free chlorine concentrations in reaction solutions were

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measured with the N,N-diethyl-p-phenylenediamine (DPD) ferrous titrimetric method and the

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DPD colorimetric method according to the standard method.43 The percentage of free chlorine


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consumed through the experiments was kept below 20% in order to maintain pseudo-first order

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conditions. Negative controls for the free chlorine experiments were run in the continuous quench-

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flow system in the same manner as the free chlorine samples, but with PBS rather than free

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chlorine.

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UV254 experiments. The UV254 experimental solutions consisted of 2.4 mL of Phi6 (4-5 × 1010

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PFU mL-1) in PBS continuously stirred in 10 mL glass beakers. Samples were exposed to UV254 in

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a collimated beam reactor44 with 0.16 mW cm-2 lamps (model G15T8, Philips) that were regularly

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measured with chemical actinometry.45 The average UV254 intensity was corrected based on the

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solution absorbance at UV254 and the sample depth. After correcting for shielding, the UV254

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intensity was 0.14 mW cm-2. Samples were exposed to UV254 for 0, 5, 15, and 25 min, which

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corresponded to UV254 doses of 0, 42, 130, 210 mJ cm-2. Negative controls for the UV254

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experiments were prepared in the same manner as the experimental samples, but were stirred in

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dark to capture any background virus inactivation and biomolecule reactions.

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Virus inactivation kinetics by free chlorine and UV254 were calculated based on the Chick-

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Watson model:46

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𝐶 ln # & = −𝑘𝐷 𝐶%

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where 𝐶 is the infectious titer (PFU mL-1), 𝐶% is the mean initial infectious titer (PFU mL-1), 𝑘 is

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the inactivation rate constant (L mg-1 s-1 or cm2 mJ-1), and 𝐷 is the free chlorine concentration (mg

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L-1) ´ contact time (s) or UV dose (mJ cm-2).

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RT-qPCR assays. Following the UV254 and free chlorine reactions, the viral genomes were

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extracted by QIAamp viral RNA mini kits (Qiagen). The Phi6 dsRNA genome consists of three

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segments designated as small (S), medium (M), and large (L) based on their relative sizes. Three

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primer sets for the Phi6 genome were designed and tested individually, each targeting a different


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genome segment. The sum of the three amplicons (~1500 bp) covered approximately 10.5% of the

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Phi6 genome (Table S1). The extracted viral genomes were mixed with 10 mM forward primer

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and 10 mM reverse primer at a ratio of 10:1.5:1.5 (v/v/v) in Tris-EDTA buffer (10 mM Tris-HCl,

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0.1 mM EDTA, pH 7.7), and then heated at 99 °C for 5 min, and quickly chilled on ice for 5 min

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before mixing with RT-qPCR reagents. RT-qPCR reactions were prepared in 96-well plates

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(Eppendorf) and conducted in duplicates on a Mastercycler ep RealPlex 2 system (Eppendorf) with

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Gotaq OneStep RT-qPCR kits (Promega). The 20-µL RT-qPCR reactions consisted of 10 µL 2´

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qPCR master mix, 0.4 µL 50´ RT mix, 5.2 µL template-primer mixture, 4 µL 5 M Betaine (Sigma-

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Aldrich), and 0.4 µL nuclease-free water. The RT reaction was conducted at 40 °C for 15 min,

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followed by an initial PCR activation step at 95 °C for 10 min. The PCR reaction included 40

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cycles of DNA denaturation at 95 °C for 15 s, primer annealing at 59 °C for 30 s, and extension at

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72 °C for 40 s. Melting curves were conducted by increasing the temperature from 60 °C to 95 °C

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over 10 min. RNA standards used for the RT-qPCR calibration curves consisted of Phi6 genomes

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extracted from the purified stock and quantified with a Qubit Fluorometer 2.0 (ThermoFisher

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Scientific). The amplification efficiencies (mean ± standard deviation) of RT-qPCR reactions

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targeting S, M, and L genome segment were 0.82 ± 0.10, 0.81 ± 0.05, and 0.81 ± 0.07, respectively.

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The mean R2 were ≥ 0.99. The reaction kinetics of RT-qPCR target regions were modeled with

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first-order reactions:

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ln +

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The reactions of the whole genome were predicted by extrapolating RT-qPCR results from the

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~1500 bp covered by the three target regions:47

𝑁 . 0 = −𝑘1,. 𝐷 𝑁%,.


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log4% #

∑.78,9,: 𝐿. 𝑁 𝑁 . &=+ 0 ? log4% + 0 ∑.78,9,: 𝐿;

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0.05 for each ANCOVA comparison). When the RT-qPCR results were extrapolated to the entire

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genome, the approximated reaction rate constant of the Phi6 genome (0.063 ± 0.012 cm2 mJ-1) was

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not significantly different from the rate constant of Phi6 inactivation (0.067 ± 0.005 cm2 mJ-1) (P

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> 0.05). This suggests that genome reactions drive UV254 inactivation of the enveloped virus Phi6.

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Although this type of analysis has not been previously reported for enveloped viruses, our finding

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is consistent with previous research on nonenveloped viruses.12,30,56,60 A comparison with the per

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base reaction rate constants measured here with those reported previously suggests that the dsRNA

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genome of Phi6 (2.4´ 10-6 cm2 mJ-1 base-1) are more resistant to UV254 than the dsDNA genome of

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adenovirus (11 ´ 10-6 cm2 mJ-1 base-1)61 and the ssRNA genome of MS2 (24 ´ 10-6 cm2 mJ-1 base-

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1

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repaired by the host cell;12 a similar repair mechanism has not been reported for the RNA viruses.

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We note that using RT-qPCR to estimate genome damage misses a fraction of the RNA

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modifications that can be detected by mass spectrometry.44 Work on the photolysis of MS2 by

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UV254, however, demonstrated that a single-hit inactivation model was appropriate for damage

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detected with RT-qPCR; in other words, every modification in MS2 RNA that causes virus

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inactivation can be detected by RT-qPCR.47 Our ongoing work aims to better characterize the

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chemistry and biological impact of RNA and DNA modifications detected by reverse

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transcriptases, polymerases, and mass spectrometry.

).56 It is worth noting that in the case of adenovirus, the modified bases detected by PCR can be


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Reactions in Phi6 proteins. The Phi6 virion contains 11 distinct proteins that are assembled

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into three layers, including viral membrane proteins (P3, P6, P9, P10, P13), nucleocapsid proteins

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(P5, P8), and polymerase complex proteins (P1, P2, P4, P7) (Figure S6).38 The functions of these

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proteins in the Phi6 life cycle have been reviewed in previous literature and are briefly described

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in the SI (Figure S6).38 Our LC-MS/MS method was capable of detecting a total of 184 pairs of

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and P2 (Figure S7). The repeated poor coverage of P6 and P2 was likely due to the low number of

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P6 and P2 protein copies in the viral particles (Figure S7).

N- and 15N-labelled Phi6 peptides. Protein coverage was over 60% for all proteins except for P6

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Free chlorine reactions. We tracked Phi6 protein degradation over the first 2-log10 Phi6

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inactivation in order to model the peptide reactions with first-order kinetics. Free chlorine reacted

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with all Phi6 peptides with reaction rate constants ranging from 0.41 to 6.3 L mg-1 s-1 (Figure 3;

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Table S6). As expected, the most reactive peptides contained Met or Cys residues (Table S6).

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Despite the similar reactivity of Met and Cys in the free amino acid form (Table S5), the most

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reactive Cys-containing peptide C257-F267 in Phi6 P4 (2.8 ± 0.3 L mg-1 s-1) reacted slower than

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the most reactive Met-containing peptide D448-R463 (6.3 ± 0.9 L mg-1 s-1) in Phi6 P1 (Table S6).

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Furthermore, the rate constants of peptides containing Met varied. For example, in the Phi6 P1,

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the rate constant of M198-K208 (1.1 ± 0.1 L mg-1 s-1) was approximately 3 ´ smaller than that of

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peptide L53-Y66 (3.1 ± 0.5 L mg-1 s-1) and 4 ´ smaller than that of peptide M209-K215 (4.5 ± 0.7

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L mg-1 s-1), despite the three peptides having spatially adjacent Met residues (Figure S8). The

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variation in reactivity of peptides containing Met and Cys is likely related to the accessibility of

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free chlorine to the amino acids.30 Indeed, the Cryo-EM model (PDB ID: 5muu) of Phi6 suggest

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that in the P1 complex, the dimethyl sulfide of M198 in M198-K208 is protected by surrounding

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amino acid residues, whereas the M65 in L53-Y66 and M209 in M209-K215 have higher solvent-


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accessible surface areas (SASA, Figure S8). It is also worth noting that oxidized Met residues were

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the only products in Phi6 proteins detected following 2-log10 inactivation by free chlorine (Figure

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S9).

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We initially hypothesized that the increased susceptibility of Phi6 to free chlorine inactivation

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compared to nonenveloped viruses, such as MS2, was due to reactions in the membrane proteins.

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These proteins play critical roles in the early steps of virus infection, and are located on the

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outermost layer of the viral particle (Figure S6). In fact, our results showed that in Phi6, some

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membrane proteins (e.g., P6, P9, P10, P13) reacted slower than the nucleocapsid proteins (e.g.,

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P8) and polymerase proteins (e.g., P1, P2, P4) (Figure 3; Table S6). This suggests that free chlorine

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molecules readily penetrate the lipid membrane and react with proteins in the nucleocapsid and

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polymerase complex. Similar findings were reported in bacteria, where the non-dissociated HOCl

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molecules could penetrate the negatively-charged bacterial membrane to react with intracellular

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structures.62

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The 8 most reactive peptides found in Phi6 proteins P3, P8, P1, P2, and P4 had rate constants

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that were comparable to the Phi6 inactivation rate constant (Figure 4; Table S6), forming Met

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oxidations as the main products (Figure S9). Consequently, one or several of these protein

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reactions may drive Phi6 inactivation by free chlorine. Given the protein reactivity and the Phi6

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life cycle, Phi6 inactivation may be due to the direct interruption of the ability to bind host cell

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(P3) or to penetrate plasma membrane (P8).63,64 Alternatively, damage to proteins P2, P4, and P7

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may indirectly inactivate Phi6 by causing changes in the vial structure.48,64,65

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Peptides in Phi6 proteins were more reactive with free chlorine than peptides in the

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nonenveloped MS2 proteins. The two most reactive peptides D448-R463 (6.3 ± 0.9 L mg-1 s-1) and

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I678-R697 (5.9 ± 1.0 L mg-1 s-1) in Phi6 were approximately 150´ more reactive than the fastest


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reacting peptide S373-R388 in the MS2 A protein (0.033 L mg-1 s-1).56 The marked discrepancies

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in reactivity of Phi6 and MS2 peptides may be due to the relative solvent accessibilities of their

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reactive amino acids.30 The average SASAs of the M456 and M680 residue in the Phi6 peptide are

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95 Å2 and 77 Å2, respectively, as calculated by YASARA software.66 Unfortunately, the SASA

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cannot be estimated for the Met in the most reactive MS2 peptide due to the fact that the crystal

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structure of the MS2 A protein has not been resolved.

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UV254 reactions. All Phi6 peptides reacted following UV254 exposure, but much less than they

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reacted with free chlorine at the same levels of inactivation. Peptide concentrations decreased by

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less than 50% following 4.2-log10 Phi6 inactivation (Figure 3), with rate constants ranging from

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0.0009 to 0.0048 cm2 mJ-1 (Table S6). Similar reaction rate constants were reported for peptides

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in bacteriophage MS2, fr, and GA proteins with UV254.30,56 Certain peptides in membrane protein

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P3 and RNA polymerase P2 reacted with faster kinetics, likely due to the presence of one or more

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UV-reactive amino acids in their sequences, including Trp (W), Tyr (Y), or Phe (F) (Table S6).67

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Indirect photoreactions with nucleic acids may also play a role in the enhanced photoreactivity of

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certain viral peptides as reported in MS2.68

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Reactions in Phi6 lipids. Phi6 lipid membranes consist of glycerophospholipids, including

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phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) compounds at a

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molar ratio of 58:38:4.39 Here, we measured the relative abundances of the eight most prevalent

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PE and PG compounds in Phi6 membranes.

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Free chlorine reactions. The PE and PG relative concentrations did not decrease significantly

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over 9.1-log10 Phi6 inactivation by free chlorine (Figure S10; P > 0.05 for all tests). A number of

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products were detected in the LC-MS spectra, including monochloramine products of PE

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(16:0/16:1) and PE (18:1/16:1) (Figure 5). The peak intensities of PE monochloramine products


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increased with increasing chlorine contact time, although at the highest inactivation level (i.e., 9.1-

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log10 Phi6 inactivation), the product peak intensities were still three orders of magnitude smaller

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than the parent PE peaks (Figure 5). The low PE monochloramine product concentration within

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the Phi6 inactivation timeframe was not surprising given that the reported rate constant for this

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reaction (1.8 ´ 104 M-1s-1) is three orders of magnitude lower than the rate constants for free

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chlorine reacting with Met and Cys residues.69 Other lipid products were detected following free

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chlorine treatment, with peak intensities no greater than 1% of the parent compound peak

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intensities (Figure 5). The chemical compositions and structures of these products did not

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correspond to commonly reported lipid oxidation products, such as lipid hydroperoxides and

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chlorohydrins.70,71

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UV254 reactions. Statistically significant reductions in the relative concentrations of the eight

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major lipid compounds were not detected following UV254 doses resulting in 8.5-log10 Phi6

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inactivation (Figure S10; P > 0.05 for all tests). Likewise, no major products were detected in the

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LC-MS spectra of treated samples following UV254 treatment (Figure 5). Previous research on

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UV254 reactions with the membrane of VSV suggest that the lipid envelope do not protect VSV

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from inactivation.72

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The lipid compositions of eukaryote enveloped viruses are more diverse than those of

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bacteriophage Phi6. Influenza virus membranes, for example, contain not only PE and PG, but

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also cholesterol and phosphatidylserine (PS).40 The kinetics of these lipids with HOCl, however,

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are not markedly faster than the lipids in the Phi6 membrane.73,74 In summary, based on our Phi6

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lipid results and what has been reported for other lipids found in virus membranes, we anticipate

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that reactions in membrane lipids do not drive enveloped virus inactivation by free chlorine or

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UV254.


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Environmental implications. Enveloped viruses are often assumed to be more susceptible

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than nonenveloped viruses to inactivation in the environment, but mechanistic descriptions of their

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differing inactivation mechanisms are lacking in the literature. Our preliminary work with

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enveloped virus Phi6 sheds light on how a model enveloped virus reacts with chemical oxidants

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and UV radiation. We found that Phi6 was 30´ more susceptible to free chlorine inactivation than

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the commonly studied nonenveloped virus MS2. Our work suggests that unlike MS2, the overall

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Phi6 particle reactivity with free chlorine is driven more by protein reactions than by genome and

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lipid reactions. Free chlorine reactions in the most reactive Phi6 peptides were orders of magnitude

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faster than reactions in the most reactive MS2 peptides, specifically for Phi6 peptides that contain

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solvent-accessible Met and Cys residues. Consequently, the relatively high number of solvent-

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accessible Met and Cys residues in the Phi6 proteins may be responsible for its fast inactivation

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kinetics with free chlorine. In contrast to chlorine, UV254 inactivates Phi6 primarily by reacting

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with the genome. This is consistent with previous research on nonenveloped viruses. It is therefore

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unlikely that enveloped viruses are more susceptible than nonenveloped viruses to direct

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photolysis via sunlight or UV disinfection processes.

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Looking beyond this initial comparison of enveloped Phi6 with nonenveloped MS2, this work

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raises a number of hypotheses about virus reactivity and inactivation that can be tested with

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additional viruses. For example, future work should explore whether proteins in enveloped viruses

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are generally more susceptible to oxidants than proteins in nonenveloped viruses, and if this is the

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reason that enveloped viruses tend to be more susceptible to inactivation by chemical oxidants.

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Related to this, research should explore the link between the presence of solvent-accessible

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reactive amino acids in viral proteins with virus inactivation by oxidants. UVC inactivation should

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be tested with additional enveloped viruses that contain various genome sizes and genome types.


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Possible enveloped viruses to study in the future include vesicular stomatitis virus and avian

429

influenza viruses. These are animal viruses with particle diameters and genome types that differ

430

from Phi6 and can be propagated to stocks with high titers. This last point is important for cases

431

in which researchers wish to apply the LC-MS/MS methods described in this study. Finally,

432

enveloped viruses in open air may undergo reactions and inactivation mechanisms similar to

433

viruses in water. Future studies should aim to compare aqueous virus reactivity with aerosolized

434

virus reactivity.

435

Enveloped viruses are structurally diverse, ranging in size, envelope composition, genome

436

type, and shape. These variations are likely reflected in a range of reactivities and inactivation

437

mechanisms. That being said, the structure and composition of the Phi6 are not especially unique

438

amongst the enveloped viruses. We are therefore confident that this study on Phi6 reactivity with

439

free chlorine and UV254 will be a valuable benchmark for future studies on enveloped virus fate in

440

disinfecting processes.

441 442

ACKNOWLEDGEMENTS

443

This study was funded by National Science Foundation (NSF) #1351188. YY was supported

444

through the Barbour Scholarship and Rackham International Student Fellowship at the University

445

of Michigan. We thank Dr. Michael Dodd at the University of Washington for his help with the

446

continuous quench-flow system, Dr. Rudy J. Richardson at the University of Michigan for his

447

instructions on how to use the YASARA software, and lab technician Tom Yavaraski for his efforts

448

to maintain the Q Exactive Orbitrap high resolution mass spectrometry.

449 450

SUPPORTING INFORMATION


20

Page 21 of 31

451


The Supporting Information is available free of charge on the ACS Publications website.

452

Additional materials and methods on protein digestion, 15N-metabolic labelling, peptide

453

limit of quantification, lipid extraction; figures of SDS-PAGE of purified Phi6; data from

454

control experiment on Phi6 survival in Tris-HCl quenched chlorine solution; lab-scale

455

continuous flow quench system; lipid calibration curves; data from experiment on Phi6

456

resistance to free chlorine after storage; Phi6 structure; Phi6 protein coverage by LC-

457

MS/MS; Cryo-EM structure of Phi6 P1, relative abundances of peptides containing

458

methionine oxidations; relative abundances of Phi6 lipids following exposure to free

459

chlorine and UV254; tables of Phi6 primers and amplicon sizes; equipment settings for

460

peptide and lipid LC-MS/MS methods; reaction rate constants for Phi6 RT-qPCR regions

461

and entire genome by free chorine and UV254; the proportion of reactive bases in Phi6 and

462

MS2 genomes; the number of reactive amino acids in Phi6 proteins; Phi6 peptides and

463

reaction rate constants by free chlorine and UV254.

464 465 466 467 468


21


FC

Contact time (s) 0.0 0.3 0.6

2

4

6

8

UV dose (mJ cm-2)

10 12

0

100

150

200

250

10-2

ns

10-3

C / C0

C / C0

469 470 471 472 473 474 475 476 477 478 479 480 481 482 483

100

10-1

ns

10-2

10-5

50

UV

100

10-1

10-4

Page 22 of 31

**

10-3 10-4 10-5

10-6

10-6

10-7

10-7

FC- or UV-treated φ6

Control

Figure 1. Inactivation of Phi6 by 2 mg L-1 free chlorine (FC) and UV254 (UV). Data includes n ³ 5 replicates for each chlorine contact time and n = 3 replicates for each UV254 dose. Student’s unpaired t-tests were used to determine statistical differences of Phi6 inactivation (C/C0) by free chlorine at two contact times. ** indicates P < 0.01, and thus that Phi6 inactivation was significantly different at the two time points; ns indicates Phi6 inactivation was not significantly different at the two time points (P > 0.05).


22

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A

Contact time (s) 2

4

6

8

UV dose (mJ cm-2) 10

12

1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4

0

Ni / N0, i

Ni / N0, i

0

10-2

10-4

10-8

100

100

10-2

10-4

10-6

10-8

10-2

N / N0

N / N0

UV

C / C0 10-6

10-4

10-4 10-6

10-6

484 485 486 487 488 489 490 491 492

250

Regions on L

Regions on M

10-2

10-8

200

0.5

C / C0 100

150

0.3

Regions on S

100

100

0.6

0.4

FC

0.3

B

50

1.2 1.1 1.0 0.9 0.8 0.7

FC

1:1

10-8

UV

1: 1

Figure 2. Phi6 genome reactions when the viruses were reacted with 2 mg L-1 free chlorine (FC) and UV254 (UV). A: Reactions in three ~500 bp regions (Ni/N0,i) as measured by RT-qPCR with respect to chlorine contact time and UV254 doses. Data includes n ³ 2 replicates for each chlorine contact time and n = 3 replicates for each UV254 dose; B: Reactions in the entire Phi6 genome (N/N0). This data was extrapolated from the three RT-qPCR regions presented in A, and was reported with respect to virus inactivation (C/C0) as measured by plaque assays.


23


mean log10 inactivation

0 mg s/L

0.6 FC mg s/L

1.2 mg s/L

0 mJ/cm2

43 UV mJ/cm2

130 mJ/cm2

0 (±0.04)

1.1 (±0.47)

2.3 (±0.41)

0 (±0.09)

1.7 (±0.07)

4.2 (±0.03)

Page 24 of 31

1.0

P3 0.8 Membrane P6 0.6

P9 P10 P13

P / P0

P8

0.4

Nucleocapsid P5

0.2

0

P1

Uncovered Peptide

Polymerase Complex

P2

P4

493 494 495 496 497 498 499

P7

Figure 3. Heatplot of Phi6 protein peptide reactions following Phi6 exposure to free chlorine (FC) and UV254 (UV). Each row in the heatplot represents one peptide. Peptides were arranged based on their sequential order in proteins, and the uncovered peptides are shown in grey. Peptide concentrations (P/P0) in this heatplot were averaged from 3 independent experiments. Detailed information of peptide sequences, reaction rate constants and standard errors are provided in Table S6.


24

Page 25 of 31


C / C0 100

P / P0

100

10-1

10-2

10-3

Membrane

100

10-1

10-2

10-3

Nucleocapsid

100

10-1

10-2

10-3

Polymerase complex

10-1

10-2 53-66 LWEVGKGNIDPVMY (P1)

10-3 188-203 MDWEGSAVADAYAAIR (P3)

500 501 502 503 504 505 506 507 508 509 510 511 512

47-82 SNPVVAGLTLAQIGSTGYD AYQQLLENHPEVAEMLK (P8)

209-215 MLQATFK (P1) 448-463 DYALDRDPMVAIAALR (P1) 678-697 IEMIGTTGIGASAVHLAQSR (P1) 15-24 AQMLFANNIK (P2) 282-296 AADTHMPSMDRPTSM (P4)

Figure 4. Decay of the 8 most reactive Phi6 peptides (P/P0) by free chlorine with respect to virus inactivation (C/C0). Data below the LC-MS/MS limit of quantification is shown in grey.


25


FC

UV 850

Control

800

800

750

750

m/z

700

700

650

650

600 850

Control

600 850

5

2.3-log10

6 7

800

1.8-log10

log10 (Peak Intensity)

m/z

850

800

750

m/z

m/z

8

750

700

700

650

650

600

600 850

5

9.1-log10

7

800 1

800

8

2

750

750

m/z

9 10 3

700

8.5-log10

6

m/z

850

Page 26 of 31

700

4

650

650

600

600 400

513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531

500

600

700

Retention time (seconds)

800

400

500

600

700

800

Retention time (seconds)

Figure 5. Phi6 lipids data collected by LC-MS before and after treatments of free chlorine (FC) and UV254 (UV). Arrows identify specific lipid products [M-H]- following free chlorine treatment, including the following accurate masses (1) 775.513; (2) 779.464; (3) 710.477; (4) 684.461; (5) 846.588; (6) 820.572; (7) 804.541; (8) 778.525; (9) 748.469 (monochloramine of PE(16:0/16:1)); (10) 722.454 (monochloramine of PE(18:1/16:1)).


26

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Reactivity of Enveloped Virus Genome, Proteins, and Lipids with Free

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