Glutathione-stabilized fluorescent gold nanoclusters vary in their

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Glutathione-stabilized fluorescent gold nanoclusters vary in their influences on the proliferation of pseudorabies virus and porcine reproductive and respiratory syndrome virus Yanli Bai, Yanrong Zhou, Huabing Liu, Liurong Fang, Jiangong Liang, and Shaobo Xiao ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00386 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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ACS Applied Nano Materials

Glutathione-stabilized fluorescent gold nanoclusters vary in their influences on the proliferation of pseudorabies virus and porcine reproductive and respiratory syndrome virus

Yanli Bai, *,†,§

†

†,§,‡

Yanrong Zhou, †,#,‡ Huabing Liu,

†, §

Liurong Fang, †,# Jiangong Liang,

†

and Shaobo Xiao *, ,#

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,

Wuhan 430070, P.R. China §

College of Science, Huazhong Agricultural University, Wuhan 430070, P.R. China

#

College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070,

P.R. China

Corresponding Authors *E-mail: [email protected] [email protected] ‡

These authors contributed equally.

*These authors jointly supervised this work.

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ABSTRACT: Gold nanoclusters (Au NCs) are widely used in biological imaging and anti-tumor treatment due to their excellent cell membrane permeability, good fluorescence properties, and high biocompatibility. However, their effects on viruses are still largely unknown. Here, pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) were used respectively as the models of DNA virus and RNA virus to investigate the influences of glutathione (GSH)-stabilized fluorescent Au nanoclusters on viruses by plaque assay, indirect immunofluorescence assay, quantitative real-time PCR (RT-qPCR) assay and Western blot assay. The experimental data indicated that Au NCs selectively inhibited proliferation and protein expression of PRRSV, but not that of PRV. Mechanistically, Au NCs directly inactivated PRRSV and blocked viral adsorption, but showed no effect on viral genome replication. These findings prompt the possibility to develop Au NCs as an effective specific antiviral nanomaterial against RNA virus infection in the future.

KEYWORDS: gold nanoclusters, porcine reproductive and respiratory syndrome virus, pseudorabies virus, proliferation, selective inhibitory effect


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1. INTRODUCTION Functional nanoparticles (NPs) have attracted increasing attention in the field of nanomedicine.1 Many kinds of nanoparticles with good antiviral activities have been found, such as quantum dots (QDs),2 carbon dots (CDs),3 silver nanoparticles (Ag NPs),4-8 gold nanoparticles (Au NPs),9-11 glycofullerenes,12 and graphene oxide13. Deokar et al. reported that sulfonated magnetic nanoparticles functionalized with reduced graphene oxide (SMRGO) is an effective and rapid antiviral agent for herpes simplex virus type 1 (HSV-1).14 Illescas et al. demonstrated that multivalent glycosylated fullerene significantly prevents Ebola virus infection in the sub-nanomolar range.15 However, there are two common drawbacks for current antiviral nanoparticles: (i) the majority of nanoparticles release heavy metals (besides gold) with cytotoxicity; (ii) the antiviral properties of most nanoparticles are of no selectivity and specificity. For example, Ag NPs inhibit propagation of both RNA viruses (human immunodeficiency virus and influenza virus)4, 7 and DNA viruses (adenoviruses, HSV, and monkey pox virus).5-6, 8 Gold nanoclusters (Au NCs) are widely used as a novel type of fluorescent nanomaterials in biology for their good biocompatibility and biosecurity.16 Previous studies have revealed their morphological properties17-19 and anti-tumor effects,20-24 as well as their potential application in cell imaging.25-26 For instance, Au NCs acted as drug carriers for the therapy of cancer.20 Chen et al. established a dual targeting function luminescent gold nanocluster (AuNC-cRGD-Apt) for tumor imaging and deep tissue therapy.22 However, to our knowledge, the influence of Au NCs on virus has not yet



been studied. Many RNA viruses, such as Ebola virus, hepatitis C virus and influenza virus, pose great threats to human health and livestock industry,27-29 suggesting the necessity to develop selective inhibitors of RNA virus.30-31 In this study, both porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded RNA virus of Arteriviridae family,32 and pseudorabies virus (PRV), a double-stranded DNA virus of the Herpesvirinae family that causes porcine Aujeszky’s disease,33-35 are important pathogens that have caused serious loss to pig industry worldwide,36-37 were selected as models of RNA or DNA virus to investigate the effect of glutathione (GSH)-stabilized Au NCs on RNA virus (PRRSV) and DNA virus (PRV). Mechanistic studies revealed a selective antiviral effect of Au NCs on PRRSV proliferation through directly inactivating viruses and interfering with viral adsorption, but not that of PRV. These data have paved the way for further research on Au NCs as a specific inhibitor against RNA virus infection.

2. EXPERIMENTAL SECTION 2.1. Synthesis of Au NCs. Regents used in this study are listed in Table S1. All glassware was immersed in freshly prepared aqua regia solution for 12 h, then washed thoroughly with ultrapure water. Au NCs were prepared as reported by Zhang et al.38 with some modifications. Briefly, 15.0 mL His solution (0.10 M) and 5.0 mL HAuCl4 solution (0.010 M) were added in a small glass bottle and reacted at 25 °C in dark for 2 h. After that, 5.0 mL GSH solution (0.12 M) was mixed with the above Au NC precursor


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solution, and Au NCs were obtained after reaction at 25 °C in dark for 12 h. The concentration of AuNCs was calculated according to the Au atom concentration of AuNCs solution, which was detected according to the method from Corpuz et al.39 2.2. Cytotoxicity Assay. MTT assay was used to detect the cytotoxicity of Au NCs as reported previously.40 Briefly, PK-15 or MARC-145 cells were incubated with Au NCs (0, 56, 112, 225, 450 and 900 µM) in 96-well plates. After incubation for 2 h, the supernatant was replaced with fresh DMEM (2% FBS), followed by incubation separately for 24 h or 48 h. Cells were then treated with MTT and maintained for 4 h. After removal of MTT reagent, the formazan precipitate was dissolved with dimethyl sulfoxide, followed by gentle shaking of the plates for10 min. Finally, the viability was calculated by comparison with control cells without exposure to Au NCs according to absorbance at 570 nm. 2.3. Virus propagation. GFP-PRV, a kind of recombinant PRV that expresses green ﬂuorescent protein, were propagated in PK-15 cells for 24 hours post infection (hpi). Similarly, PRRSV were propagated in MARC-145 cells for 48 hpi. Next, the cells were frozen at -80 °C. The progeny viruses were released three times via freeze-thaw, and kept at -80°C for further use.41 2.4. Antiviral activity assay. Main processes of virus replication cycle include attachment, entry, genome replication, protein synthesis and exocytosis. To explore the exact step(s) Au NCs target for antiviral effect, the antiviral activity assay with four different treatment conditions were performed by changing the incubation sequence of Au NCs and viruses with the cells using a previously reported method.42 (1) virus pre-



treatment assay: 0.1 MOI (multiplicity of infection) GFP-PRV was mixed with Au NCs (450 μM) for 1 h, then PK-15 cells were infected with the mixture for another 2 h; (2) co-treatment assay: 0.1 MOI GFP-PRV was mixed with Au NCs (450 μM) and immediately infected PK-15 cells for 2 h; (3) cell pre-treatment assay: PK-15 cells were pretreated with Au NCs (450 μM) for 1 h, followed by infection with 0.1 MOI GFPPRV; (4) cell post-treatment assay: PK-15 cells were infected with 0.1 MOI GFP-PRV for 2 h in the absence of Au NCs, and then treated with Au NCs (450 μM) for another 2 h. The effect of Au NCs on PRV infection was further evaluated using confocal microscopic analysis. Similarly, PRRSV (MOI = 0.1) was treated with Au NCs (450 μM) and infected MARC-145 cells as described above and the influence of Au NCs on PRRSV proliferation was further observed with indirect immunofluorescence assay and confocal microscopic analysis at 36 hpi. 2.5. Indirect immunofluorescence assay and confocal microscopic analysis. The PRRSV in MARC-145 cells were observed using indirect immunofluorescence assay as reported in our previous work.3 In brief, MARC-145 cells were treated as described in “antiviral activity assay” and fixed at 36 hpi. Primary antibody against nucleocapsid (N) protein of PRRSV and Alexa Fluor 594-conjugated donkey anti-mouse IgG were used to detect fluorescence signals of the infected cells. As for GFP-PRV, PK-15 cells were treated as described in “antiviral activity assay” and fixed at 24 hpi. Confocal microscope was used to observe the fluorescence signals of GFP-PRV infected cells.


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2.6. Plaque assay. PRV (MOI=0.1) were treated as described in “virus pre-treatment assay” and collected separately at 6, 9, 12, 18 and 24 hpi. The virus titers of PRV were detected by plaque assay as reported by Luo et al.43 Briefly, PK-15 cells were cultured in 6-well plates for 24 h until 95% confluence. Next, virus samples infected the PK-15 cells for 1 h. Then, the cells were covered with the mixture of low melting point agarose and 2 × DMEM with 1% penicillin-streptomycin and 3% FBS.44 The results were obtained from three parallel experiments after 2-3 days post infection at 37 °C. Likewise, virus titers of PRRSV were obtained as described above. 2.7. Western blot assay. PRRSV (MOI=0.1) were treated as described in “virus pretreatment assay” and then harvested at 36 hpi. The expression of PRRSV N protein was analyzed with Western blot assay by using mouse anti-N antibody.45 2.8. RT-qPCR assay. MARC-145 cells were treated with Au NCs (450 μM) after infection with 0.1 MOI PRRSV for 6 h. The negative-sense PRRSV RNA at 7, 8, 9 and 10 h post Au NCs incubation was detected by the reported methods.46 The primer specific to 5’-terminal region of PRRSV genomic was as follows: 5’GACGTATAGGTGTTGGCTC-3’. The primers used in qPCR assay were: sense 5’GCATTTGTATTGTCAGGAGC-3’

and

antisense

5’-

AGCAGTGCAACTCCGGAAG-3’. Gene expression was analyzed as previously described.47, 48 2.9. Statistical analysis. Cytotoxicity and antiviral activity of Au NCs were analyzed with one-way analysis of variance. Results were shown as the mean ± standard deviation (SD) of three independent experiments. P < 0.05 or P < 0.001 stand for



statistical significance.

3. RESULTS AND DISCUSSION 3.1. Characteristics of Au NCs. The apparatus used to characterize Au NCs in our study are lists in Table S2. The UV-Vis absorption and fluorescent spectra of Au NCs were shown in Figure 1A. The absorption spectra were detected between 230 and 650 nm, with a small peak observed near 402 nm. It can be seen that no obvious absorption spectra were detected at >550 nm, which is consistent with previously reported Au NCs.39 In addition, a strong fluorescent emission peak was observed at 502 nm, with a strong green fluorescence, indicating discrete excitation-emission bands because of inter-band transitions.39, 49 The fluorescence lifetimes of Au NCs were also indicated in Figure S1, which was calculated to be 5.20 ± 0.05 ns. The quantum yield of Au NCs was 6.8% according to a reported procedure.39 The transmission electron microscopy (TEM) image (Figure S2) of Au NCs indicated that the average diameter was 1.5± 0.5 nm. The results of dynamic light scattering (DLS) (Figure 1B) revealed the good dispersity of Au NCs, and the hydrodynamic diameter distribution was 2.5 ± 0.7 nm, which is consistent with the results of TEM.


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Figure 1. (A)The UV-Vis absorption and fluorescence spectra of Au NCs, and (B) hydrodynamic diameter distribution of Au NCs from DLS. The fluorescent excitation wavelength was 425 nm. Figure 2A shows the Fourier transform infrared (FT-IR) spectroscopy of the asprepared Au NCs. Stretch vibrations of C-OH at 3418 cm-1, C-H at 2945 cm-1, C-NH at 1506 cm-1 and C=O at 1631 cm-1 represented the characteristic absorption bands of GSH and His,9-10, 49-50 and the stretch vibrations of S-H at 2620 cm-1 showed the characteristic peaks of Au-S. The full range X-Ray Photoelectron Spectrometer (XPS) analysis (Figure 2B) showed five evident peaks at 84.0, 163.0, 285.0, 400.1, and 530.6 eV, which could be attributed to Au4f, S2p, C1s, N1s and O1s, respectively. The Au4f core level spectra are shown in Figure 2C, with the peaks at 84.0 and 87.6 eV, corresponding to Au4f7/2 and Au4f5/2, respectively.51 The spectrum of S2p (Figure 2D) revealed the existence of S22-, S2-, and S0/Sn 2-,52 demonstrating the Au-S bond on the surface of Au NCs.39 The peaks of C1s (Figure 2E) at 284.4, 285.1, 285.8 and 287.5 eV were assigned to C-C, C-N, C-O and O=C-N, respectively.53-55 N-(C)3, C-N-C and C=N were confirmed according to the three fitted peaks at 400.6, 399.3 and 398.1 eV in N1s spectrum (Figure 2F) .53, 56 C-O (531.6 eV) and C=O (530.6 eV) were also proved by the XPS spectrum of O1s (Figure 2G).57 The presence of C-NH and Au-S from FT-IR and XPS suggested that histidine and glutathione are on the surface of the Au NCs, and COOH and NH2 are the main functional groups of Au NCs.



3.2. Cytotoxicity of Au NCs. In previous reports, Au NCs have been found to have low cytotoxicity on various types of cells.58-60 For instance, Zhang et al. observed that ATII and A549 cells still have high viability under the treatment of GSH-stabilized Au NCs even at the concentration of 1000 μM.39 To assess the potential cytotoxicity of Au NCs, the MTT assays were conducted in both PK-15 and MARC-145 cells. As shown in Figure 3, after incubation with different concentrations (0, 56, 112, 225, 450 and 900 µM) of Au NCs, the relative viabilities of the two types of cells were both more than 95% when treated with 450 µM Au NCs, and close to 94 and 91% at 900 µM, respectively. These results indicated low toxicity of Au NCs and good biocompatibility with different types of cells. Hence, 450 μM was selected for the following studies.


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Figure 2. FT-IR spectra (A) and XPS spectra of Au NCs (B), Au4f (C), S2p (D), C1s (E), N1s (F) and O1s(G).



Figure 3. Cytotoxicity of Au NCs analyzed by MTT assay.

3.3. Influence of Au NCs on viral proliferation. To evaluate the effect of Au NCs on propagation of DNA or RNA virus, PRV or PRRSV were treated with Au NCs as described in the “antiviral activity assay” in experimental section and the results were analyzed using indirect immunofluorescence assay and confocal microscopic analysis. As depicted in Figure 4A, Au NCs showed no effect on the proliferation of PRV under all four treatment conditions. However, Au NCs significantly prevented PRRSV infection when PRRSV was pre-incubated with Au NCs before infection (Figure 4B, the third line and Figure S3) or when MARC-145 cells were co-treated with PRRSV and Au NCs (Figure 4B, the fourth line), but did not affect the proliferation of PRRSV when MARC-145 cells were pre-incubated with Au NCs before PRRSV infection (Figure 4B, the fifth line) or when PRRSV infected MARC-145 cells prior to Au NCs incubation (Figure 4B, the sixth line). These results demonstrated that Au NCs selectively inhibit the infection of PRRSV (a model of RNA virus) through directly


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inactivating viruses and interfering with viral adsorption, but not that of PRV (a model of DNA virus).

Figure 4. Antiviral activity assay of Au NCs against PRV infection (A) or PRRSV infection (B) with indirect immunofluorescence assay and confocal microscopic analysis. The first row: cells (PK-15 and MARC-145 cells) without any treatment; the second row: virus infected cells; the third row: virus incubated with Au NCs for 1 h before infecting cells; the fourth row: cells co-treated with virus and Au NCs; the fifth row: Au NCs was added after virus infection; the sixth row: Au NCs was added before virus infection.

3.4. The effect of Au NCs on viral infection measured with plaque assay. To further confirm the inactivation effect of Au NCs on viral infection, plaque assay, a classic method to investigate viral titers, was used here as described in experimental section. As displayed in Figure 5A, the virus titer of Au NCs-treated PRV (Au NCsPRV) resembled that of control PRV, indicating that Au NCs have no obvious influence



on propagation of PRV. In addition, we found that the titers of both Au NCs-PRV and control PRV could reach approximately 108 pfu/mL at 18 hpi, suggesting that the infectivity of PRV was well preserved under the treatment of 450 μM of Au NCs, which was extremely vital for the potential application of Au NCs in the intracellular trafficking of labeled virus. On the contrary, as shown in Figure 5B, the relative titers of Au NCs-PRRSV showed a remarkable reduction relative to the PRRSV control. Interestingly, the antiviral effect of the Au NCs on the later stage (36 and 48 hpi) of virus multiplication was more significant than that on the early stage (6, 12 and 24 hpi). The results from plaque assay further proved the effective antiviral effect of Au NCs on PRRSV, rather than PRV, which were in accordance with the data form confocal microscopic analysis.

Figure 5. Propagation of PRV (A) and PRRSV (B) with or without 450 μM of Au NCs.

3.5. The effect of Au NCs on PRRSV N protein. PRRSV N protein, as an abundant viral protein during infection, has been found associated with the pathogenicity of PRRSV.61 The expression levels of N protein were detected through a Western blot assay as described in experimental section to further validate the direct inactivation activity of Au NCs on PRRSV infection. As depicted in Figure 6, Au NCs were found


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to significantly inhibit the N protein synthesis of PRRSV. Collectively, the results of indirect immunofluorescence assay, plaque assay and western blot assay verified that Au NCs can significantly inhibit the proliferation of RNA virus (PRRSV), but have no significant influence on the progeny virus infection of PRV in PK-15 cells.

Figure 6. Western blot analysis of PRRSV N protein with or without the treatment of Au NCs.

3.6. Preliminary exploration of potential mechanism for selective antiviral effect of Au NCs. Currently, numerous nanoparticles have been found to play antiviral roles by targeting various stage(s) of virus infection. For example, Barras et al. reported that boronic acid-modified carbon nanodots can inhibit HSV-1 infection by interfering with virus entry,62 while mercaptoethane sulfonate-modified gold nanoparticles could act as an effective anti-HSV-1 agent via a cell-surface-receptor heparan sulfate-dependent cell entry mechanism.9 In addition, Ziem et. al also found that the polyvalent 2D, which was based on polyglycerol sulfate-functionalized graphene sheets, can act as efficient entry inhibitors for enveloped viruses, such as PRV and African swine fever virus .63 In the present study, Au NCs were found to have specific antiviral effect on PRRSV infection by directly inactivating viruses and interfering with viral adsorption. However, the detailed mechanism for the selective antiviral activity of Au NCs remains to be elucidated in future work.



To investigate whether the virus-infected cell type is related with the discrepancy in antiviral properties, we tested the effects of Au NCs on PRV propagation in MARC145 cells as described in experimental section using plaque assay. As presented in Figure S4, Au NCs also had no obvious influence on PRV proliferation in MARC-145 cells. These data eliminated the involvement of cell type in the different antiviral effect of Au NCs on PRV and PRRSV propagation. Considering that the replication process of DNA viruses (PRV) mainly happens in nucleus, while that of RNA viruses (PRRSV) mostly occurs in cytoplasm,64 cellular imaging of Au NCs was performed for a preliminary investigation of whether the localization of Au NCs is similar to that of PRRSV. PK-15 and MARC-145 cells were incubated separately with 450 μM of Au NCs for 2 h, then the cells were observed with an excitation wavelength of 405 nm. From Figure S5, it can be seen that Au NCs are strictly localized to cytoplasm, rather than nucleus, of both PK-15 and MARC-145 cells. Therefore, we speculate that Au NCs might specifically inhibit the proliferation of RNA viruses based on the similar localization. To verify the hypothesis, the effect of Au NCs on PRRSV genome replication was tested as described in a previous paper.46 As shown in Figure S6, no significant differences were observed in PRRSV negative RNA levels before and after treatment of Au NCs, suggesting that Au NCs have no noticeable effect against the genome replication process of PRRSV. Meanwhile, the results imply that the localization of Au NCs to the cytoplasm might not be the predominant factor for the selective antiviral activity of Au NCs on RNA viruses.


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Additionally, we notice that the concentration of Au NCs used in our study is beyond the general bioavailability of tissues and may not be suitable for in vivo application. However, PRRSV was reported to spread through close contact with fomites, such as faeces and urine from infected pigs, and aerial transmission,65 suggesting the potential of Au NCs as a novel PRRSV inhibitor via inactivating viruses in the environment. Furthermore, coupling virus-specific agents, such as antibodies and aptamers, on the surface of NPs is an effective strategy to improve the antiviral activity and reduce the working concentration of NPs, enabling Au NCs to be applied in future as a safe antiviral nanomaterial in vivo.

4. CONCLUSIONS In summary, we systematically investigated the antiviral activities of Au NCs using PRRSV and PRV as DNA and RNA

virus models. Through indirect

immunofluorescence assay, Au NCs were found to have selective antagonistic effect against the propagation of RNA virus (PRRSV) by directly inactivating PRRSV and interfering with viral adsorption, but not that of DNA virus (PRV) at a noncytotoxic concentration. The results was further confirmed by the Western blot assay, plaque assay and RT-qPCR assay, which means Au NCs may play an important role in inhibiting RAN viruses as a novel antiviral agent, and facilitate a better understanding of the relationship between Au NCs and viruses.

ASSOCIATED CONTENT



Supporting Information. Supplementary data of Au NCs including: apparatuses and regents used in our study; fluorescence decay curves; TEM image; inactivation activity on PRRSV infection using indirect immunofluorescence assay; the effect on PRV infection in MARC-145 cells; fluorescence microscopy images; the effect on PRRSV genome replication.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J Liang) *E-mail: [email protected] (S Xiao) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We gratefully acknowledge the financial support from National Key Research and Development Program (2016YFD0500105), and National Natural Science Foundation of China (31490602, 31372439).


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Au NCs selectively inhibit PRRSV infection. 35x18mm (600 x 600 DPI)


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Glutathione-stabilized fluorescent gold nanoclusters vary in their

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