Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus

Abstract

Possible mechanisms that lead to inactivation of feline calicivirus (FCV) by cold atmospheric-pressure plasma (CAP) generated in 99% argon-1% O2 admixture were studied. We evaluated the impact of CAP exposure on the FCV viral capsid protein and RNA employing several cultural, molecular, proteomic and morphologic characteristics techniques. In the case of long exposure (2 min) to CAP, the reactive species of CAP strongly oxidized the major domains of the viral capsid protein (VP1) leading to disintegration of a majority of viral capsids. In the case of short exposure (15 s), some of the virus particles retained their capsid structure undamaged but failed to infect the host cells in vitro. In the latter virus particles, CAP exposure led to the oxidation of specific amino acids located in functional peptide residues in the P2 subdomain of the protrusion (P) domain, the dimeric interface region of VP1 dimers, and the movable hinge region linking the S and P domains. These regions of the capsid are known to play an essential role in the attachment and entry of the virus to the host cell. These observations suggest that the oxidative effect of CAP species inactivates the virus by hindering virus attachment and entry into the host cell. Furthermore, we found that the oxidative impact of plasma species led to oxidation and damage of viral RNA once it becomes unpacked due to capsid destruction. The latter effect most likely plays a secondary role in virus inactivation since the intact FCV genome is infectious even after damage to the capsid.

Fig 1. Schematic diagram of the plasma jet including sample treatment and electrical and gas inputs.

Fig 2. CAP exposure effect on FCV infectivity.

A) Infectious FCV titer before and after exposure to CAP for 15, 30, 60, and 120s. B) Inverted microscope image (magnification power 40 X) of CRFK cells infected with CAP-unexposed and CAP-exposed (15s exposure) FCV at different post-infection times up to 5 days. Serial of 10-fold dilutions from triplicate samples of CAP-exposed and unexposed FCV were prepared separately. Each dilution was inoculated in CRFK cell monolayers (three wells per dilution) and then incubated at 37°C under 5% CO₂ for 5 days. The number of positive wells showing CPE was recorded for each sample and the infectious virus titer was calculated by the Karber method [30]. Columns are the average value of triplicate samples and the error bars represent the standard deviations.

Fig 3

Transmission electron microscopic images of FCV (magnification 200,000 X): A) CAP-unexposed virus; arrow indicates virus particles). B) CAP-exposed for 15s (black arrows refer to distorted viral particles; red arrows to debris of plasma-affected viral particles; and blue arrow to intact virions).

Fig 4. Quantification of capsid-destruction as a function of CAP-exposure time.

A) Diagram explaining the principle of RT-qPCR coupled with ethidium monoazide (EMA-RT-qPCR). B) The titer of capsid-intact FCV after 15 and 120 s exposures to CAP as compared to control (CAP-unexposed). The data are average of triplicate measurements and error bars represent standard deviation. Labels on top of columns refer to the significance of RNA concentration as compared to control of each sample [*: Statistically significant (P≤0.05)]. C) The agarose gel patterns of EMA-coupled conventional RT-PCR products generated from viral RNA obtained from 15s and 120s CAP-exposed and unexposed FCV samples. Aliquots (100 μl) of 15s and 120s CAP-exposed FCV were mixed with 5μg EMA/100 μl followed by incubation at 4°C for 30 min in dark. Samples were then exposed to light (using a 650W halogen bulb) for 5 min in ice bath followed by viral RNA extraction. Quantification of PCR-reactive and PCR-nonreactive RNAs in control and CAP-exposed samples was carried out by RT-qPCR. Conventional RT-PCR reactions were carried out using RNA extracted from control and CAP-exposed samples to compare them qualitatively based on agarose gel patterns of a region of viral RNA nucleotide.

Fig 5. One dimensional SDS-PAGE (4–15% gradient gel) image of CAP-exposed FCV proteins (15 s and 120 s vs. control).

The gel was stained with Imperial Protein Stain (Thermo Scientific, Rockford, IL). Two gel regions from each lane were excised; upper region from approximately 50–74 KD and a lower region from approximately 27–43 KD.

Fig 6

Representative LC-MS/MS annotated spectrum (top) with alignment (bottom) of: A) monoisotopic [M + 2H]2+ observed precursor 795.182 m/z matched to peptide LAAIVVPPGVRPVQSTSM(+16)LQYPHVLFDAR PEAKS -10logP score 46.17. B) monoisotopic [M + 2H]²⁺ observed precursors 616.9438 m/z matched to peptide HFDFNQETAGW(+30)ITPR, PEAKS -10logP score 39.07. Theoretical b- and y-type fragment ion types matched to experimental product ion peaks are labeled (spectrum copied and pasted from PEAKS® Studio 7.0). The identified b and y ions are mapped onto the primary sequence and demonstrate modifications in the identified peptides. The peptide scores for the set of representative peptides have a false discovery rate of 0.5%.

Fig 7. Space-filling model of the structure of the major capsid protein (VP1) and the complete FCV capsid.

A) Location of all domains constructing the VP1protein. The yellow colored regions represent the whole NTA domain (I); the whole S domain (II); the whole P1 subdomain (III); and the whole P2 subdomain (IV). B) Location of oxidized peptide residues (yellow colored) among NTA domain (I)*; S domain (II); P1 subdomain (III); P2 subdomain (IV). C) Location of all VP1 domains within the whole FCV capsid. The yellow colored regions represent the whole NTA domain (I); the whole S domain (II); the whole P1 subdomain (III); and the whole P2 subdomain (IV). D) Location of oxidized peptide residues (yellow colored) among NTA domain (I)*; S domain (II); P1 subdomain (III); P2 subdomain (IV). Images were created by Cn3D software version 4.3 using crystal structure of FCV [41] available at: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=83065 * No yellow color since there was no oxidized peptide residue detected in NTA domain.

Fig 8. Crystal structure of A/B dimer of the major capsid protein (VP1) using warm model.

A) The crystal structure of A/B dimer showing the dimer interface region, the domains of VP1 (S, P1, P2, and NTA domains), and the location of oxidized peptide residues (in yellow color) within the crystal structure of A/B dimer. B) The crystal structure of the oxidized peptide residues only (yellow colored) virtually separated from the entire A/B dimer. Images were created by Cn3D software version 4.3 using the crystal structure of FCV [41] available at: http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=83065.

Fig 9. FCV genome integration.

A) RT-PCR products of RNA isolated from CAP-unexposed FCV. B) RT-PCR products of RNA isolated from CAP-exposed FCV for 2 min (capsid-packed RNA). C) RT-PCR products of unpacked FCV-RNA exposed for 15 s to CAP. Eleven primer pairs (Table 1) covering the entire viral genome were used in RT-PCR to amplify the viral genome in a total of 11 DNA amplicons.

Fig 10. Exposure time dependent effect of CAP on viral RNA.

A) Agarose pattern of RT-PCR products from capsid-packed RNA exposed to CAP (sequence targeted by primer set #5). B) Agarose pattern of RT-PCR products from unpacked viral RNA exposed to CAP (sequence targeted by primer set #5). Agarose gel concentration was 1.2% that could separate amplicon sizes from 50–10,000 bp.