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. 2025 Jan 22;97(10):5480–5487. doi: 10.1021/acs.analchem.4c04852

Viral Membrane-Targeting Amphipathic Helical Peptide-Based Fluorogenic Probes for the Analysis of Infectious Titers of Enveloped Viruses

Yusuke Sato †,‡,§,*, Yusaku Hatanaka †, Yoshitaka Sato ∥, Kota Matsumoto †, Shion Osana ⊥, Ryoichi Nagatomi ⊥, Seiichi Nishizawa †,*
PMCID: PMC11923947  PMID: 39840494

Abstract

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Enveloped viruses have caused the majority of epidemics and pandemics over the past decade. Direct sensing of virus particles (virions) holds great potential for the functional analysis of enveloped viruses. Here, we explore a series of viral membrane-targeting amphipathic helical (AH) peptide-based molecular probes for the assessment of infectious titers of the human coronavirus 229E virus (HCoV-229E). The M2-protein-derived AH peptide is identified as a strong binder for HCoV-229E, and its conjugate with Nile Red, M2-NR, exhibits fluorogenic response upon selective binding to the viral membrane of HCoV-229E. We demonstrate that the response of M2-NR toward the HCoV-229E virus enables the rapid, simple, and reliable assessment of the infectivity of HCoV-229E. In addition, the present fluorescence assay for infectivity analysis is applicable to various kinds of enveloped virus including influenza A virus, herpes simplex virus-1, and lentivirus.

Emerging and re-emerging viral diseases pose a serious threat to global public health. A large fraction of recent viral outbreaks (e.g., the COVID-19 pandemic) has been caused by enveloped viruses characterized by a lipid bilayer (envelop) surrounding the capsid.1 Besides their importance as target analytes for human health, recent studies revealed that the enveloped viruses could serve as functional materials for biomedical applications including vaccines2 and gene therapies.3 Reliable analytical techniques for enveloped viruses are crucial for virology and virus-based material sciences. At present, most of the techniques rely on two detection principles, antibody-based immunoassays (e.g., Western blotting and enzyme-linked immunosorbent assay (ELISA)) and polymerase chain reaction (PCR), which can measure and quantify target viral proteins and viral genomes, respectively. While they are undoubtedly useful for virus analysis, the obtained data do not reflect the information on virus particles (virions) because of their incapability to distinguish target molecules originating from the virions or free proteins/nucleic acids in solutions.4 As a complement to these methods, direct sensing of virions holds great promise for the analysis of their functions of virus, such as infectious titers of virus.5−8

In contrast to recent efforts in advanced instrumentation such as flow virometry,5,6 less attention has been paid to molecular probes for virion analysis. We sought that fluorogenic molecular probes capable of selective binding to lipid membranes of enveloped virions would be useful for this purpose, as they would not suffer from free viral components. The measurement of infectious titers of a virus has always been a primary requirement for basic research on viral diseases as well as virus-related biotechnology. The tissue culture infectious dose 50 (TCID50) assay is widely used for the determination of the infectious titer of a virus based on the cytopathic effect (CPE) induced by virus infection in the cultured cells. However, this assay is very time-consuming (more than 5 days) and labor-intensive.9 These challenges are expected to be addressed if a titration method can be constructed using viral membrane-targeting fluorescent probes due to their capability of rapid and simple analysis.10,11 It is noted that lipophilic dyes such as long-chain dialkylcarbocyanines are well-known to show a fluorogenic response upon binding to viral membranes by simply inserting their lipophilic tail into the lipid bilayers. However, their use is limited to stain-enveloped virions for the visualization of the transport behaviors and dissection of the dynamic interactions with the host cells.12 These dyes are yet to be examined for the assessment of infectious titers of viruses.

In this work, we report viral membrane-binding amphipathic helical (AH) peptide-based fluorogenic probes as a proof-of-concept for the analysis of enveloped viruses (Figure 1). AH peptides, one of the most common motifs in lipid membrane-binding proteins,13 are known to sense the membrane curvature by the insertion of multiple hydrophobic residues in the α-helical AH peptide into the lipid packing defects that expose hydrophobic acyl tails (hydrophobic insertion).14,15 They have been recently employed to construct molecular probes targeting biological membranes.16−20 We and other groups demonstrated that AH peptides conjugated with environment-sensitive fluorophores17,18 or fluorogenic molecular rotators19 exhibited a binding-induced fluorescence response for highly curved membranes of exosomes, inherently small extracellular vesicles (EVs) (30–150 nm) produced by cells. These probes facilitated the fluorescence sensing of EVs in a ‘mix and read’ manner. Here, we considered that this class of AH peptides should serve as useful binders toward enveloped virions for the design of fluorescent probes, given that enveloped virions (typically, 100–200 nm) have highly curved membranes derived from the host cells as well. In contrast to earlier attempts of developing membrane-disrupting antiviral agents based on AH peptides,21 our focus is the construction of fluorescent probes for analyzing the functions of enveloped viruses. As there are no reports on AH peptide-based tools to this aim, we first screened a variety of peptide sequences to sieve out useful candidates for the human coronavirus 229E virus (HCoV-229E). We successfully identified an M2 protein-derived AH peptide as a strong binder to the membranes of HCoV-229E and demonstrated that the M2 peptide probe conjugated with Nile Red (M2-NR) exhibited significant fluorogenic response upon binding to HCoV-229E. Moreover, M2-NR enabled the rapid, simple, and reliable infectivity analysis of a variety of enveloped virus including HCoV-229E, influenza A virus, herpes simplex virus-1, and lentivirus. These binding and fluorescence-sensing properties were discussed based on the molecular basis to develop virion-targeting molecular tools for enveloped virus analysis.

Figure 1.

Figure 1

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Schematic illustration of AH peptide-based fluorogenic probes targeting viral membranes of the enveloped virus.

Materials and Methods

Reagents

Fmoc-protected amino acids with the L-configuration except glycine were purchased from Watanabe Chemical Industries (Hiroshima, Japan) or AAPPTec (Louisville, KY, U.S.A.). FITC-I was purchased from Thermo Fisher Scientific (Tokyo, Japan). All phospholipids (1-palmitoy-2-oleyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoy-2-oleyl-sn-glycero3-phospho-l-serine (POPS), 1-palmitoy-2-oleyl-sn-glycero-3-phosphocholine (POPC)) and cholesterol were purchased from Avanti Lipids (Alabaster, AL, USA). Nile Red (NR) derivatives (6-(9-diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic acid and 2-((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)acetic acid) were synthesized according to the literature.17,22 Recombinant adeno-associated virus-2 reference standard stocks were purchased from ATCC (Manassas, VA, USA). Other reagents were commercially available and used without further purification. Water was deionized (≥18.0 MΩ cm specific resistance) by an Elix 5 UV water purification system and a Milli-Q Synthesis A10 system (Millipore Corp., Bedford, MA), followed by filtration through a BioPak filter (Millipore Corp.) to remove RNase. In all assays, the amount of DMSO was adjusted to a final concentration <1%. Unless otherwise mentioned, all measurements were performed at 25 °C in 1 × PBS buffer solution (pH 7.4). Errors are the standard deviations obtained from three independent experiments (N = 3).

Probe Synthesis

All probes were synthesized using Biotage Initiator+ Alstra peptide synthesizer (Biotage, Uppsala, Sweden) based on Fmoc solid-phase peptide chemistry on a Rink-Amide-ChemMatrix resin (Biotage). A 1-[(1-(cyano-2-ethoxy-2-oxoethylideneamino-oxy)-dimethylamino-morpholino methylene)]methana minium hexafluorophosphate (COMU)/diisopropylethylamine (DIEA) system was employed for the coupling reaction. After completion of the elongation of all amino acid residue units, the fluorophores with reactive groups (FITC-I or 6-(9-diethylamino-5-oxo-5H-benzo [a]phenoxazin-2- yloxy)hexanoic acid) were introduced at the N-terminus. The deprotection of the peptides and the cleavage from the resin were conducted using trifluoroacetic acid/triisopropylsilane/water (95/2.5/2.5). The solution was dropped into cold diethyl ether in order to precipitate the crude peptide probe. The obtained crude product was purified by a reverse-phase HPLC system (pump, PU-2086 Plus × 2; mixer, MX 2080-32; column oven, CO-1565; detector, UV-2070 plus and UV-1570 M (Japan Spectroscopic Co. Ltd., Tokyo, Japan)) equipped with a C18 column (Inertsil ODS3 (5.0 μm particle size, 250 × 20 mm column size); GL Sciences Inc., Tokyo, Japan) using a gradient of water/acetonitrile containing 0.1% TFA (see Figure S2A). The probe was verified by MALDI-TOF-MS (Bruker Daltonics autoflex Speed-S1, Germany) (see Figure S2B and Table S2). The concentration of NR-carrying peptide probes was determined based on the molar absorption coefficient of 6-(9-(diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)-hexanoic acid at 550 nm in MeOH (ε = 30,423 cm–1 M–1).17 The concentration of FITC-carrying peptide probes was determined based on the molar absorption of FITC at 494 nm in 1 × PBS buffer (ε = 78,219 cm–1 M–1).

Preparation of Synthetic Liposomes

Synthetic liposomes were prepared by extrusion according to the literature.23 Chloroform-suspended POPC, POPE, POPS, and cholesterol were combined to form lipid mixtures of the desired molar ratio. The lipid film was obtained by drying under nitrogen gas and then hydrated overnight at 4 °C in PBS buffer (pH 7.4). The lipid suspension was subjected to five freeze–thaw cycles. The vesicles were then prepared by manual extrusion through polycarbonate membranes (Whatman, NJ, USA) with pore diameters of 100, 400, and 1000 nm using Avanti mini-extruder (Avanti lipids), which affords average diameters of 104 nm (V104), 593 nm (V593), and 903 nm (V903), respectively. The liposome size was characterized by dynamic light scattering (DLS) measurements (Zetasizer Nano-ZS, Malvern, UK).

Fluorescence Measurements

Fluorescence spectra and anisotropies were recorded on a JASCO model FP-6500 spectrofluorophotometer (Japan Spectroscopic Co. Ltd., Tokyo, Japan) with a thermoelectrically temperature-controlled cell holder. Measurements were performed using a 3 × 3 mm quartz cuvette. As for the spectral measurements, polarizers (excitation and emission polarization set to horizontal and vertical, respectively) were used in order to avoid the effect of light scattering by the vesicles.24

For the measurements with Laurdan, the excitation wavelength was set to 340 nm. The generalized polarization (GP) value for Laurdan was determined by the equation GP = (I440 – I490)/(I440 + I490), where I440 and I490 are the emission intensities of Laurdan at 440 and 490 nm, respectively.25

Circular Dischroisim (CD) Measurements

CD spectra were measured with a JASCO model J-800 spectropolarimeter equipped with a thermoelectrically temperature-controlled cell holder (Japan Spectroscopic Co. Ltd., Tokyo, Japan) using a 2 × 1 mm quartz cuvette (optical path length: 1 mm).

Cells

Cells were purchased from ATCC (Rockville, MD, USA) or the RIKEN BRC cell bank (Tsukuba, Japan). LLC-MK2 (ATCC, CCL-7) and MDCK cells (ATCC, CCL-34) were cultured in minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS and 1% penicillin–streptomycin (Pen/St). HEK293T (ATCC, CRL-3216) and Huh-7 (RIKEN BRC Cell Bank, RCB 1942) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% GlutaMAX, and 1% penicillin–streptomycin (Pen/St). Vero cells (ATCC, CCL-81) were cultured in Eagle’s minimal essential medium supplemented with 10% calf serum (CS), 1% l-glutamine, and 1% Pen/St.

Viral Stocks

The HCoV strain 229E was obtained as shown in the literature26 or purchased from ATCC (VR-740). The influenza A virus, A/California/07/2009 (H1N1) pdm09, was purchased from ATCC (VR-1894). The HSV-1 strain F was propagated in Vero cells. The CSII-CMV-MCS-IRES2-Venus vector was obtained from RIKEN BioResource Center (Wako, Japan). Lentivirus pseudotyped with VSV-G was prepared as described previously.27

Viral Infection and Collection of Virus or Viral Components

LLC-MK2 cells were cultured for 2 days in plastic 24-well plates. The cells were rinsed with phosphate-buffered saline (PBS) and then infected with the HCoV-229E virus (500 mL per well; 1.0 × 104 50% tissue culture infectious dose (TCID50)/mL; 1.0 TCID50/cell of the multiplicity of infection) for 24 h at 33 °C with 5% CO2. After incubation, 500 mL of fresh MEM medium was added to each well. The culture plates were incubated at 33 °C for 5 days, until CPEs were observed. The supernatants were collected and centrifuged at 1200 × g to remove cells. The buffer exchange to 1 × PBS buffer was performed using 100 kDa Amicon Ultra centrifugal filters (Merck Millipore, Watford, UK), and the obtained samples were stored at–80 °C until measurement.

Total RNA and total protein were extracted from virus-infected LLC-MK2 cells using the TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA). The concentration of the obtained RNA was measured by a Shimadzu UV-1900i UV–vis spectrophotometer equipped with a Nano Stick (Scinco, Seoul, Korea). The concentration of total protein was determined using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). Final concentrations of total RNA and total protein in the examination of fluorescence response of M2-NR were 0.2 and 2.6 μg/mL, respectively. We confirmed that the obtained total RNA and total protein can be detected by RT-PCR and Western blotting with anti-N-protein antibody, respectively.

Western Blotting

A 3 mL portion of the Laemmli buffer with 0.5% isopropanol was added to the viral solution and heated for 5 min at 95 °C. The denatured proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% gels and transferred onto nitrocellulose membranes using a Bio-Rad dry blotting system (Bio-Rad, Hercules, CA, USA). The membranes were blocked with EveryBlot Blocking Buffer (Bio-Rad) for 5 min at room temperature. After the membranes were washed three times with TBS buffer with 0.1% Tween-20 (TBS-T), they were incubated with rabbit antinucleocapsid protein (N protein) of HCoV-229E antibodies (0.31 μg/mL) overnight at 4 °C. The membranes were washed three times with TBS-T and then incubated with a secondary antirabbit IgG (HRP-linked antibody antibodies; 1:2000, Cell Signaling Technology, Danvers, MA, USA) for 2 h. After being washed three times with TBS-T, the membranes were immersed in a chemiluminescence reagent (Immuno Star Zeta; Fujifilm Wako Pure Chemical corporation, Tokyo, Japan) and visualized by an ImageQuant LAS 4000 (Fujifilm Wako Pure Chemical Corporation).

RT-qPCR

Viral RNA was reverse-transcribed to cDNA using a ReverTra Ace qPCR RT Master Mix with a gDNA Remover (TOYOBO, Osaka, Japan). Real-time PCR was performed on the CFX Opus 96 System (Bio-Rad, Hercules, CA, USA) using a THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, Osaka, Japan). The primer sequences used are 5′-CAGTC AAATG GGCTG ATGCA-3′ and 5′-AAAGG GCTAT AAAGA ATAAG GTATT CT-3′ for HCoV-229E.

Virus Titer Determination

The viral titer was determined by quantifying the TCID50 assay or reporter gene expression. Cells were plated in 96-well plates at a density of 5 × 103 cells/well. After 2 days, virus solutions were subjected to 10-fold serial dilutions (101–108) and inoculated onto the cells. The cells were incubated at 33 or 37 °C with 5% CO2. After 5 days, each inoculated well was evaluated for the presence or absence of viral CPE. TCID50 was calculated based on the Spearman–Karber method. For determining the titer of VSV-G LV, HEK293T cells were infected with the virus, and Venus-positive cells were counted by a fluorescence-activated cell sorter to measure the viral titer (infectious units (IFUs)/mL).

Results and Discussion

Screening of AH Peptide Sequences for HCoV-229E

We initiated the identification of AH peptide candidates targeting HCoV-229E from a repertoire of AH peptide sequences within highly curved membrane-binding proteins (Figure 2A). The AH peptide from the C-terminal of apolipoprotein A-I (ApoC) was shown to selectively and strongly bind to highly curved membranes over flat ones.17,19,28 Arf1 and p2–23 sequences were selected due to their capability to sense the membrane curvature.29,30 We also examined the M2 peptide that originated from the cytoplasmic domain of the influenza virus M2 protein, which mediates the membrane scission in the virus budding process.31 C5A and 27AH, derived from the N-terminal of the hepatitis C virus nonstructural protein 5A (NS5A), were reported to bind to several kinds of enveloped viruses.21 These peptides differ in physicochemical parameters including net charge, mean hydrophobicity, and hydrophobic moment (Figure S1 and Table S1).

Figure 2.

Figure 2

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(A) Sequences of AH peptides used for the screening of the binders toward the HCoV-229E virus. X denotes norleucine used instead of methionine in the wild sequence (Table S1). (B) Chemical structure of M2-NR. (C) Fluorescence response of the AH peptide conjugated with the NR unit (2.0 μM) for 1.7 × 109 particles/mL HCoV-229E virus. Measurements were performed in PBS buffer at 25 °C with excitation at 552 nm. Analysis, 640 nm.

We synthesized AH peptide probes labeled with an NR unit, an environment-sensitive fluorophore, at the N-terminal for the examination of HCoV-229E (Figure 2B). All peptide probes were synthesized based on the Fmoc solid-phase chemistry. The obtained crude probes were purified by a reverse-phase HPLC system, and the purified probes were verified by MALDI-TOF-MS (Figure S2 and Table S2). We used the HCoV-229E virus propagated in LLC-MK2 (rhesus monkey kidney) cells, in which mean diameters and particle concentrations were determined as 122 nm and 3.4 × 109 particles/mL, respectively, by nanoparticle tracking analysis (Figure S3). Figure 2C shows the changes in the fluorescence intensity at 640 nm of AH peptide–NR probes (2.0 μM) upon addition of 1.7 × 109 particles/mL HCoV-229E virus in a PBS buffer at 25 °C. We observed the fluorescence enhancement of all probes for the virus in a concentration-dependent manner, which can be ascribed to the partition of the NR unit by the binding of the AH peptide unit to the virus. Significantly, M2-NR (Figure 2B) demonstrated a more pronounced response, an 8.3-fold increase in the emission, compared to other NR probes carrying AH peptides including C5A and 27AH, which were previously developed as virion-binding peptides,12 indicating the strong binding of the M2 peptide for HCoV-229E. This was further supported by the examination of filter-binding assays using FITC-labeled peptides (Figure S4).29 Briefly, FITC-labeled peptides were incubated with HCoV-229E, and subsequent removal of unbound peptides was carried out through multiple washing steps utilizing 100 kDa Amicon Ultra filters. The retained fluorescence intensity of the peptides bound to the virus was assessed for the binding property of the peptides. We found that the M2 peptide displayed the largest retained fluorescence among the peptides, which was in good agreement with the results using NR probes (cf. Figure 2C). These results signify the useful binding property of the M2 peptide toward HCoV-229E. We examined the number of M2 peptides bound to HCoV-229E based on the results obtained by the filter-binding assay with FITC-labeled M2. We confirmed that the emission intensity of FITC-labeled M2 was unaffected upon binding to HCoV-229E (data not shown). Together with the fact that unbound peptides could be completely removed (more than 99.9%), the number of FITC-labeled M2 bound to one HCoV-229E particle was calculated to be 3020 based on the linear calibration curve of the FITC-labeled M2 concentration versus its fluorescence intensity. This value was almost comparable to that for another AH peptide bound to bacterial EVs.20

Fluorescence Sensing of HCoV-229E by M2-NR

Prior to this study, the examination of the M2 peptide was limited to the binding to the influenza virus.29−31 We thus characterized the binding and fluorescence-signaling functions of M2-NR (Figure 2B) for HCoV-229E in detail. Figure 3A depicts the fluorescence spectra of M2-NR in the absence and presence of HCoV-229E, measured in PBS buffer at 25 °C. The NR unit of the probe showed a weak background emission with a maximum at 662 nm. The addition of 1.7 × 109 particles/mL HCoV-229E rapidly caused significant fluorescence enhancement as well as a blue shift of the emission peak (27 nm), as observed for synthetic liposomes with sizes comparable to those of HCoV-229E virions (Figure S5A). We also observed the fluorescence enhancement of M2-NR for liposomes in a membrane-curvature-dependent manner (Figure S5A). The M2 unit was induced to fold into an α-helix structure upon binding to virion-sized liposomes (Figure S5B). Hence, the observed fluorescence response for HCoV-229E is highly likely due to the partition of the NR unit into the lipid membrane upon the hydrophobic insertion of the α-helical M2 unit into the lipid packing defects present on membranes of HCoV-229E (cf. Figure 1).31 The examination of the concentration of M2-NR revealed that 2.0 μM M2-NR provided the largest fluorescence response to HCoV-229E (Figure S6). Further experiments were conducted with 2.0 μM M2-NR.

Figure 3.

Figure 3

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(A) Fluorescence spectra of M2-NR (2.0 μM) in the absence and presence of 1.7 × 109 particles/mL HCoV-229E. The response to 1.7 × 109 particles/mL AAV-2 is also shown. (B) Effect of protease K (0.1 mg/mL) or Triton-X (0.1%) treatment on the fluorescence response of M2-NR (2.0 μM) to 1.7 × 109 particles/mL HCoV-229E. (C) Fluorescence response of M2-NR (2.0 μM) to serially diluted HCoV-229E (0–1.7 × 109 particles/mL). Excitation, 552 nm. Analysis, 640 nm. Temperature, 25 °C.

In order to gain further insights into the functions of M2-NR, we examined two kinds of NR probes carrying mutant peptides: mut1 (RLKKFGIYRFFEHGLFRS) and mut2 (RLAAKSAARFAEHGLKRG)32 (Figure S7). Mut1 corresponds to the scrambled sequence of the M2 peptide. Mut2 was designed by replacing bulky and hydrophobic residues in the hydrophobic face of the α-helix to alanine.32 The Mut1-NR probe exhibited a much weaker response (2.2-fold, Figure S7) to HCoV-229E compared to M2-NR despite the same total cationic charge. Thus, the binding of the M2 unit to HCoV-229E is not simply driven by electrostatic interactions. We observed a negligible response of the Mut2 probe possessing a poorly developed hydrophobic face in comparison with M2-NR (Figure S1, which clearly shows the crucial role of the hydrophobic residues of the α-helical M2 unit in the binding to HCoV-229E. These results are highly consistent with the recognition of lipid packing defects by the M2 unit in the NR probe.

We next examined the influence of surface proteins on the binding of M2-NR to HCoV-229E. It was found that the treatment of protease K did not result in the attenuation of the fluorescence response (Figure 3B). Apparently, M2-NR does not respond to viral proteins on the membranes such as spike proteins of HCoV-229E. Meanwhile, the fluorescence response of M2-NR clearly disappeared upon the treatment of the triton-X detergent (Figure 3B). These results thereby affirm the binding of M2-NR to the viral membrane of HCoV-229E. Notably, M2-NR exhibited no response to adeno-associated virus-2 (AAV-2) virions that are composed of a nonenveloped capsid at a particle concentration equivalent to that of the HCoV-229E virus (Figure 3A), which shows its specific targeting property toward enveloped viruses. Considering the effect of the linker length and the position of the NR unit on the fluorescence response for HCoV-229E (Figure S8), we quantified M2-NR (Figure 2B) as a fluorogenic probe for further investigation.

We examined the response of M2-NR to serially diluted HCoV-229E virus (Figure 3C), which clearly shows that the emission of the NR unit was linearly enhanced as the HCoV-229E particle concentration was increased (slope: 0.42). This yields the limit of detection (LOD) of 1.0 × 107 particles/mL based on 3 times the standard deviation of the blank. The detection sensitivity was superior by at least 1 order of magnitude to Western blotting using the anti-N protein antibody (Figure S9). It should be noted here that the fluorescence sensing by M2-NR features the minimal interference from viral proteins and viral RNAs. M2-NR exhibited negligible response to total RNAs (0.2 μg/mL) and total proteins (2.6 μg/mL) extracted from HCoV-229E virus-infected LLC-MK2 cells (Figure S10). This is attributed to the selective binding and fluorescence-signaling capability of M2-NR toward the membranes of HCoV-229E. Such a selectivity enables the avoidance of interference from these virus-derived components, which stands in sharp contrast to conventional immunoassays and PCR assays.4

We evaluated the feasibility of M2-NR for HCoV-229E analysis in a biological fluid, for which pooled human saliva spiked with HCoV-229E was examined as saliva is a useful specimen for coronavirus analysis in the clinic.33 Preliminary experiments showed that M2-NR displayed a blue-shifted and stronger emission of M2-NR in the saliva samples without HCoV-229E (Figure S11A), which would be due to the response to saliva components, including EVs. It was found that this interference from saliva components could be largely reduced by pretreatment using Amicon Ultra centrifugal filters for buffer exchange and concentration (Figure S11B). Especially, we found minimum interference for 10% saliva samples by this pretreatment procedure. Thus, HCoV-229E-spiked 10% saliva samples were subjected to the pretreatment, followed by the fluorescence sensing with M2-NR. M2-NR displayed a good fluorescence response for the spiked saliva samples (Figure S11C), where the response was dependent on the virus concentration. The LOD value was determined as 2.6 × 107 particles/mL after taking the dilution factor into account, which is comparable to that measured in PBS buffer (cf. Figure 3C). These results suggest the applicability of M2-NR for HCoV-229E analysis in saliva.

Assessment of the Infectivity of HCoV-229E by the Fluorescence Response of M2-NR

Motivated by the above findings, M2-NR was applied to the assessment of the infectivity of HCoV-229E by means of its fluorescence response. Here, we independently collected five kinds of HCoV-229E virus samples with different titers determined by the TCID50 assay. The virus particle concentration of each sample was determined based on the above-mentioned calibration curve of M2-NR response for the serially diluted HCoV-229E virus (Figure 3C). Figure 4A depicts the relationship of the virus particle concentration determined by the fluorescence assay with M2-NR against the TCID50/mL value by cell-based assays. Significantly, a good linear relationship between the two assays was obtained (R2 = 0.975). M2-NR assays provided a higher correlation with TCID50 assays in comparison with PCR assays (R2 = 0.833, Figure S12). This clearly shows that the fluorescence assay using M2-NR enables us to assess the virus titer of HCoV-229E. The infectious titer of a virus sample can thus be estimated by the M2-NR response to HCoV-229E, resulting in the calculated titer being highly consistent with that determined by the TCID50 assay (Figure 4B). Our assay exhibits the distinct advantages of simplicity and rapid analysis (within 5 min) for infectivity analysis compared to conventional TCID50 assays.

Figure 4.

Figure 4

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(A) Linear relationship between the HCoV-229E particle concentration (particles/mL) by the M2-NR response and the virus titer based on the TCID50 assay. (B) Comparison of the virus titer calculated by an M2-NR fluorescence assay and those determined by TCID50 assay; [M2-NR] = 2.0 μM.

Application to the Analysis of Various Enveloped Viruses

We next assessed whether our fluorescence assay was applicable to HCoV-229E from other host cells. Similar to the case of HCoV-229E from LLC-MK2 cells, M2-NR exhibited fluorescence enhancement upon binding to HCoV-229E propagated from Huh-7 (human hepatoma) cells (HCoV-229EHuh-7). The slope of the calibration curve was found to be 0.48 (Figure 5A), which is comparable to that from LLC-MK2 cells (slope: 0.42; cf. Figure 3C). This indicates that these virions have similar membrane properties regarding the lipid packing defects targeted by M2-NR, irrespective of the variation in host cells. The calculated infectious titer of HCoV-229EHuh-7 with M2-NR agreed with that from the TCID50 assay (Figure 5B). We further examined three kinds of enveloped viruses: influenza A virus (IAV: A/California/07/2009 (H1N1) pdm09), herpes simplex virus-1 (HSV-1: F strain), and lentivirus pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G LV). The titer values of IAV and HSV-1 were determined by the observation of CPE in cell-based assays as well (Materials and Methods). Meanwhile, the titer of VSV-LV was assessed based on fluorescent-protein-positive infected cells using flow cytometry as it does not produce obvious CPE. We calculated the titer of these viruses using a preconstructed calibration curve of M2-NR for serially diluted samples for each virus (Figure 5A). As a result, a high correlation between our assay and the cell-based assay was clearly observed (Figure 5B). Hence, the fluorescence response of M2-NR was indeed applicable to estimate the infectivity of a wide variety of enveloped viruses. It is noteworthy that even enveloped viruses without CPE can be analyzed by our assay. Our assay was demonstrated to be effective for the analysis of a single kind of enveloped virus in the samples. However, it is needed to improve the selectivity of probes toward target enveloped viruses in the case of the analysis of a mixture with another kind of enveloped virus.

Figure 5.

Figure 5

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(A) Fluorescence response of M2-NR to serially diluted virus (HCoV-229EHuh-7, IAV, HSV-1, or VSV-G LV). (B) Comparison of the virus titer (HCoV-229EHuh-7 (TCID50/mL), IAV (TCID50/mL), HSV (TCID50/mL), or VSV-G LV (IFU/mL)), calculated by the M2-NR fluorescence assay and those determined by cell-based assays; [M2-NR] = 2.0 μM.

Interestingly, we found distinct differences in the linear slopes of the calibration curves for the examined enveloped viruses (Figure 5A). M2-NR exhibited much larger responses for HSV-1 and VSV-G LV compared to HCoV-229E (slope: HSV-1, 1.7; VSV-G LV, 1.3). On the other hand, the smallest response was observed for IAV (slope: 0.091). These results suggest the variation in the lipid packing defects on the membranes for these viruses. It has been recognized that the occurrence of lipid packing defects is associated with the membrane curvature and the lipid composition.34,35 In our case, the former would be ruled out given that these virions have comparable sizes (diameters: 120–130 nm) based on nanotracking analysis (Figure S13). Thus, the latter is highly likely to be responsible for the observed difference in the lipid packing defects, whereas there are no reports on systematic lipidomics analysis for these virions. In order to gain further insights into the viral membrane properties, the level of lipid order was estimated by Laurdan, a membrane environment-sensitive fluorophore (Figure S14).36 Laurdan undergoes emission spectrum shifts according to the exposure of the dye to polar media, where the spectral shifts can be quantified by the GP. The degree of lipid order (GP value) of the viral membranes is found to be in the order of IAV (0.45) > HCoV-229E (0.38–0.35) > HSV-1 (0.2) > VSV-G LV (0.15), which matches the order of the M2-NR fluorescence response. The obtained results are plausible as lipid orderliness is suggested to be correlated with the lipid packing defects.37 As the lipid packing defects on the membranes control the adsorption of membrane-binding proteins,13−15 these finding would contribute to better understanding the functions and behaviors of the virions during the infection process.

Conclusions

In summary, we developed a new class of viral membrane-targeting AH peptide probes for the analysis of enveloped viruses. The M2 peptide identified here serves as a strong binder for the HCoV-229E virus, and its conjugate with the NR unit, M2-NR, exhibited a binding-induced fluorescence response upon selective binding to the enveloped membranes of HCoV-229E. M2-NR was applicable to the fluorescence sensing of HCoV-229E spiked in saliva samples, which indicates the potential for virus detection in biological fluids. Furthermore, we demonstrated the proof-of-concept that M2-NR facilitated the assessment of infectious titers of various enveloped viruses. Once the calibration curve is established for target enveloped viruses, the present assay allows the rapid, simple, and reliable analysis of the virus infectivity without any sophisticated instruments.5−7 These features should be beneficial for the routine determination of infectious titers in virology research. To the best of our knowledge, this is the first report on molecular probes for the assessment of the infectious titers of an enveloped virus. In addition, we expect that the present assay for the infectious titers holds great potential for application toward point-of-care testing to evaluate the infectivity of biological virus samples. As for the application of the diagnosis of viral infection toward clinical use, we envision that the present assay can be used in parallel with PCR to enhance the assay stringency. As demonstrated here, our infectivity analysis displayed high consistency with the cell-based TCID50 assay (HCoV-229E, IAV, and HSV-1) and flow cytometry (VSV-LV), which shows that the M2-NR fluorescent response should mainly originate from binding to virus particles. On the other hand, we note that the binding of M2-NR to infected-cell-producing EVs would contribute to the observed fluorescence response.4,6 It should be noted that almost all studies of molecular probes targeting virions did not account for EV contamination in virus samples, presumably due to the technical difficulty in the separation of virions from EVs.38 As preliminary experiments to address the effect of coexisting EVs, we found that the titer estimation for HCoV-229E by M2-NR was not largely affected by the presence of uninfected LLC-MK2 cell-producing EVs (Figure S15). However, we could not rule out the possible response to EVs from HCoV-229E-infected cells at this time as the amount of such EVs and the difference in membrane properties between uninfected and infected-cell-producing EVs are not clear. Given recent reports that the infected-cell-producing EVs can aid the virus infection,39−41 we need to address the effects of such contaminants on M2-NR functions for virus analysis in the next step. Improvement of the functions of the probe is also needed for the analysis of enveloped viruses in the serum, containing more complicated components than saliva. The development of probes with improved binding affinity and fluorescence-signaling properties is necessary for the analysis of viruses with low titers. Overall, the obtained results strongly show the unique and useful functions of AH peptide-based probes in the field of virology and related biotechnology. We hope that this work will open up a new avenue for designing useful molecular tools for enveloped virus research.

Acknowledgments

We thank Prof. Hitoshi Kasai and Dr. Ryuju Suzuki for the DLS measurements. We also thank the Biomedical Research Unit of Tohoku University Hospital for technical support. This work was supported by JST PRESTO (Grant No. JPMJPR19H4 to Yusuke Sato), FOREST (Grant No. JPMJFR236J to Yusuke Sato), CREST (Grant No. JPMJCR19H4 to Yoshitaka Sato), and JSPS KAKENHI (Grant Nos. 21K18207 and 23H00297 to Seiichi Nishizawa).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c04852.

  • Peptide information, probe purification and characterization, size distribution of HCoV-229E virus, filter-binding assay, response to synthetic liposomes, effect of the M2-NR concentration, response of M2 mutants, Western blot, response to total RNA and total protein, application to virus detection in saliva, correlation between virus determined by RT-qPCR and the virus titer based on TCID50 assay, size distribution of other viruses, fluorescence response of Laurdan, effect of EVs from uninfected cells on the analysis (PDF)

The authors declare no competing financial interest.

Supplementary Material

ac4c04852_si_001.pdf (656.7KB, pdf)

References

  1. Yoon B. K.; Jeon W.-Y.; Sut T. N.; Cho N.-J.; Jackman J. A. Stopping Membrane-Enveloped Viruses with Nanotechnology Strategies: Toward Antiviral Drug Development and Pandemic Preparedness. ACS Nano 2021, 15, 125–148. 10.1021/acsnano.0c07489. [DOI] [PubMed] [Google Scholar]
  2. Bhuket P. R. N.; Luckanagul J. A.; Rojsitthisak P.; Wang Q. Chemical modification of enveloped viruses for biomedical applications. Integr. Biol. 2018, 10, 666–679. 10.1039/C8IB00118A. [DOI] [PubMed] [Google Scholar]
  3. Lundstrom K. Viral Vectors in Gene Therapy. Diseases 2018, 6, 42–62. 10.3390/diseases6020042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Renner T. M.; Tang V. A.; Burger D.; Langlois M. A. Intact Viral Particle Counts Measured by Flow VIrometry Provide Insight into the Infectivity and Genome Packaging Efficiency of Moloney Murine Leukemia Virus. J. Virol. 2020, 94, e01600-19 10.1128/JVI.01600-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Heider S.; Metzner C. Quantitative real-time single particle analysis of virions. Virol. 2014, 462–463, 199–206. 10.1016/j.virol.2014.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Zamora J. L. R.; Aguilar H. C. Flow virometry as a tool to study viruses. Methods 2018, 134–135, 87–97. 10.1016/j.ymeth.2017.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Niu Q.; Ma L.; Zhu S.; Li L.; Zheng Q.; Hou J.; Lian H.; Wu L.; Yan X. Quantitative Assessment of the Physical Virus Titer and Purity by Ultrasensitive Flow Virometry. Angew. Chem., Int. Ed. 2021, 60, 9351–9356. 10.1002/anie.202100872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Liu Y.; Potts J. L.; Bloch D.; Nian K.; McCormick C. A.; Fanari O.; Rouhanifard S. H. Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers. ACS Sens. 2023, 8, 2563–2571. 10.1021/acssensors.3c00239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hochdorfer D.; Businger R.; Hotter D.; Seifried C.; Solzin J. Automated, Label-free TCID50 Assay to Determine the Infectious Titer of Virus-based Therapeutics. J. Viorol. Methods 2022, 299, 114318 10.1016/j.jviromet.2021.114318. [DOI] [PubMed] [Google Scholar]
  10. Kobayashi H.; Ogawa M.; Alford R.; Choyke P. L.; Urano Y. New Strategy for Fluorescent Probe Design in Medical Diagnostic Imaging. Chem. Rev. 2010, 110, 2620–2640. 10.1021/cr900263j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kozma E.; Kele P. Fluorogenic Probes for Super-Resolution Microscopy. Org. Biomol. Chem. 2019, 17, 215–233. 10.1039/C8OB02711K. [DOI] [PubMed] [Google Scholar]
  12. Liu S.-L.; Wang Z.-G.; Xie H.-Y.; Liu A.-A.; Lamb D. C.; Pang D.-W. Single Particle Tracking: From Theory to Biophysical Applications. Chem. Rev. 2020, 120, 1936–1979. 10.1021/acs.chemrev.9b00692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Has C.; Das S. L. Recent Developments in Membrane Curvature Sensing and Induction by Proteins. Biochim. Biophsy. Acta Gen. Subj. 2021, 1865, 129971 10.1016/j.bbagen.2021.129971. [DOI] [PubMed] [Google Scholar]
  14. Drin G.; Antonny B. Amphiathic Helices and Membrane Curvature. FEBS Lett. 2010, 584, 1840–1847. 10.1016/j.febslet.2009.10.022. [DOI] [PubMed] [Google Scholar]
  15. Giménez-Andrés M.; Copič A.; Antonny B. The Many Faces of Amphipathic Helices. Biomolecules 2018, 8, 45. 10.3390/biom8030045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Murayama T.; Masuda T.; Afonin S.; Kawano K.; Takatani-Nakase T.; Ida H.; Takahashi Y.; Fukuma T.; Ulrich A. S.; Futaki S. Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells. Angew. Chem. Int. Ed. 2017, 56, 7644–7647. 10.1002/anie.201703578. [DOI] [PubMed] [Google Scholar]
  17. Sato Y.; Kuwahara K.; Mogami K.; Takahashi K.; Nishizawa S. Amphipathic helical peptide-based fluorogenic probes for a marker-free analysis of exosomes based on membrane-curvature sensing. RSC Adv. 2020, 10, 38323–38327. 10.1039/D0RA07763A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kawano K.; Yokoyama F.; Kawamoto J.; Ogawa T.; Kurihara T.; Futaki S. Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media. J. Mol. Biol. 2020, 432, 5876–5888. 10.1016/j.jmb.2020.09.009. [DOI] [PubMed] [Google Scholar]
  19. Ohira K.; Sato Y.; Nishizawa S. Self-Assembly and Disassembly of Membrane Curvature-Sensing Peptide-Based Deep-Red Fluorescent Probe for Highly Sensitive Sensing of Exosomes. ACS Sens. 2023, 8, 522–526. 10.1021/acssensors.2c02498. [DOI] [PubMed] [Google Scholar]
  20. Kawano K.; Kumasaka K.; Yokoyama F.; Kawamoto J.; Ogawa T.; Kurihara T.; Matsuzaki K. Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains. Biophys. Chem. 2023, 299, 107039 10.1016/j.bpc.2023.107039. [DOI] [PubMed] [Google Scholar]
  21. Jackman J. A. Antiviral Peptide Engineering for Targeting Membrane-Enveloped Viruses: Recent Progree and Future Directions Biochim. Biophys. Acta Biomembr. 2022, 1864, 183821 10.1016/j.bbamem.2021.183821. [DOI] [PubMed] [Google Scholar]
  22. Vurgun N.; Nitz M. Highly Functionalized β–Cyclodextrins by Solid-Suported Synthesis. Chem.—Eur. J. 2018, 24, 4459–4467. 10.1002/chem.201800028. [DOI] [PubMed] [Google Scholar]
  23. Saludes J. P.; Morton L. A.; Coulup S. K.; Fiorini Z.; Cook B. M.; Beninson L.; Chapman E. R.; Fleshner M.; Yin H. Multivalency Amphifies the Selection and Affinity of Bradykinin-derived Peptides for Lipid Nanovesicles. Mol. BioSyst. 2013, 9, 2005–2009. 10.1039/c3mb70109c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ladokhin A. S.; Jayasinghe S.; White S. H. How to Measure and Analyze Tryptophan Fluorescence in Membranes Properly and Why Bother ?. Anal. Biochem. 2000, 285, 235–245. 10.1006/abio.2000.4773. [DOI] [PubMed] [Google Scholar]
  25. Orlikowaska-Rzeznik H.; Krok E.; Chattopadhyay M.; Lester A.; Piatkowski L. Laurdan Discerns Lipid Membrane Hydration and Cholesterol Content. J. Phys. Chem. B 2023, 127, 3382–3391. 10.1021/acs.jpcb.3c00654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sasaki S.; Osana S.; Kubota T.; Yamaya M.; Nishimura H.; Nagatomi R.; Kaneko T. Human Coronavirus Inactivation by Atomospheric Pressure Helium Plasma. J. Phys. D: Appl. Phys. 2022, 55, 295203. 10.1088/1361-6463/ac6a8c. [DOI] [Google Scholar]
  27. Suzuki T.; Sato Y.; Okuno Y.; Goshima F.; Mikami T.; Umeda M.; Murata T.; Watanabe T.; Watashi K.; Wakita T.; Kitagawa H.; Kimura H. Genome-wide CRISPR Screen for HSV-1 Host Factors Reveals PAPSS1 Contributes to Heparan Sulfate Synthesis. Commun. Biol. 2022, 19, 694. 10.1038/s42003-022-03581-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tanaka M.; Takamura Y.; Kawakami T.; Aimoto S.; Saito H.; Mukai T. Effect of Amino Acid Distribution of Amphipathic Helical Peptide Derived from Human Apolipoprotein A-I on Membrane Curvature Sensing. FEBS Lett. 2013, 587, 510–515. 10.1016/j.febslet.2013.01.026. [DOI] [PubMed] [Google Scholar]
  29. Martyna A.; Bahsoun B.; Badham M. D.; Srinivasan S.; Howard M. J.; Rossman J. S. Membrane Remodeling by the M2 Amphipathic Helix Drives Influenza Virus Membrane Scission. Sci. Rep. 2017, 7, 44695. 10.1038/srep44695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jensen M. B.; Bhatia V. K.; Jao C. C.; Rasmussen J. E.; Pedersen S. L.; Jensen K. J.; Langen R.; Stamou D. Membrane Curvature Sensing by Amphipathic Helices: A Single Liposome Study using α-synuclein and Annexin B12. J. Biol. Chem. 2011, 286, 42603–42614. 10.1074/jbc.M111.271130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martyna A.; Gómez-Llobregat J.; Lindén M.; Rossman J. S. Curvature Sensing by a Viral Scission Protein. Biochemistry 2016, 55, 3493–3496. 10.1021/acs.biochem.6b00539. [DOI] [PubMed] [Google Scholar]
  32. Jung Y.; Kong B.; Moon S.; Yu S.-H.; Chung J.; Ban C.; Chung W.-J.; Kim S.-G.; Kweon D.-H. Envelope-Deforming Antiviral Peptide Derived from Influenza Virus M2 Protein. Biochem. Biophys. Res. Commun. 2019, 517, 507–512. 10.1016/j.bbrc.2019.07.088. [DOI] [PubMed] [Google Scholar]
  33. Yazawa S.; Yamazaki E.; Saga Y.; Itamochi M.; Inasaki N.; Shimada T.; Oishi K.; Tani H. Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19. Sci. Rep. 2023, 13, 8893. 10.1038/s41598-023-35915-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vanni S.; Hirose H.; Barelli H.; Antonny B.; Gautier R. A Sub-Nanometre View of How Membrane Curvature and Composition Modulate Lipid Packing and Protein Recruitment. Nat. Commun. 2014, 5, 4916–4925. 10.1038/ncomms5916. [DOI] [PubMed] [Google Scholar]
  35. Liu J.; Bu B.; Crowe M.; Li D.; Diao J.; Ji B. Membrane Packing Defects in Synaptic Vesicles Recruit Complexin and Synuclein. Phys. Chem. Chem. Phys. 2021, 23, 2117–2125. 10.1039/D0CP03546G. [DOI] [PubMed] [Google Scholar]
  36. Gaus K.; Zech T.; Harder T. Visualizing Membrane Microdomains by Laurdan 2-Photon Microscopy. Mol. Membr. Biol. 2006, 23, 41–48. 10.1080/09687860500466857. [DOI] [PubMed] [Google Scholar]
  37. Tripathy M.; Srivastava A. Lipid Packing in Biological Membranes Governs Protein Localization and Membrane Permeability. Biophys. J. 2023, 122, 2727–2743. 10.1016/j.bpj.2023.05.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tang V. A.; Renner T. M.; Fritzsche A. K.; Burger D.; Langlois M. A. Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry. Sci. Rep. 2017, 7, 17769. 10.1038/s41598-017-18227-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sato Y.; Yaguchi M.; Okuno Y.; Ishimaru H.; Sagou K.; Ozaki S.; Suzuki T.; Inagaki T.; Umeda M.; Watanabe T.; Fujimuro M.; Murata T.; Kimura H. Epstein-Barr Virus Tegument Protein BGLF2 in Exosomes Released from Virus-Producing Cells Facilitates de novo Infection. Cell Commun. Signal. 2022, 20, 95. 10.1186/s12964-022-00902-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tey S. K.; Lam H.; Wong S. W. K.; Zhao H.; To K.K-W.; Yam J. W. P. ACE2-Enriched Extracellular Vesicles Enhance Infectivity of Live SARS-CoV-2 Virus. J. Extracell. Vesicles 2022, 11, e12231 10.1002/jev2.12231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peng Y.; Yang Y.; Li Y.; Shi T.; Luan Y.; Yin C. Exosome and Virus Infection. Front. Immnol. 2023, 14, 1154217 10.3389/fimmu.2023.1154217. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

ac4c04852_si_001.pdf (656.7KB, pdf)

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