key: cord-0883975-gzfiedm7 authors: Chen, Daiwei; Lu, Shengsheng; Yang, Guang; Pan, Xiaoyan; Fan, Sheng; Xie, Xi; Chen, Qi; Li, Fangfang; Li, Zhonghuang; Wu, Shaohua; He, Jian title: The seafood Musculus senhousei shows anti-influenza A virus activity by targeting virion envelope lipids date: 2020-04-17 journal: Biochem Pharmacol DOI: 10.1016/j.bcp.2020.113982 sha: 8426098934fae609b10cb4d354de1d48b407e9d1 doc_id: 883975 cord_uid: gzfiedm7 Abstract Marine environments are known to be a new source of structurally diverse bioactive molecules. In this paper, we identified a porphyrin derivative of Pyropheophorbide a (PPa) from the mussel Musculus senhousei (M. senhousei) that showed broad anti-influenza A virus activity in vitro against a panel of influenza A viral strains. The analysis of the mechanism of action indicated that PPa functions in the early stage of virus infection by interacting with the lipid bilayer of the virion, resulting in an alteration of membrane-associated functions, thereby blocking the entry of enveloped viruses into host cells. In addition, the anti-influenza A virus activity of PPa was further assessed in mice infected with the influenza A virus. The survival rate and mean survival time of mice were apparently prolonged compared with the control group which was not treated with the drug. Therefore, PPa and its derivatives may represent lead compounds for controlling influenza A virus infection. Musculus senhousei is a traditional Chinese seafood that is widely distributed all over the Pacific coast including the USA, Australia, Japan, and China, among other countries 1 . This mussel is reported to live in the region ranging from intertidal to shallow subtidal zones at the depth of approximately 30 m, and it is tolerant of low salinity and low oxygen levels during its life span of ca. two years (http://www.exoticsguide.org/musculista_senhousia). The outside shell of M. senhousei is smooth and shiny with a yellow-green color and can grow to a maximum length of 35 mm, while its interior is purplish-gray. Analyses of the habitat and growth conditions indicate that M. senhousei is a passive filter-feeding shellfish. Thus, in addition to a small number of protozoa, the main component of its food is diatoms, which belong to 20 different genera 2 . Marine environments have long been viewed as a major reservoir of bioactive molecules that have the potential to be developed as therapeutic drugs 3 . In our continuous search for anti-influenza A viral compounds from natural sources 4-6 using A/FM-1/1/47 (H1N1) mouse adjustment strain, A/Puerto Rico/8/34 (H1N1) with NA-H274Y mutation, and A/Aichi/2/68 (H3N2) were propagated in 9-day-old chick embryo at 37 °C, and the allantoic fluid containing the above viruses were stored at −80 ℃ and quantified in a 50% tissue culture infectious dose (TCID 50 ) test until needed. The fresh mussels (40 kg) were grounded, and repeatedly extracted for at least three times in 95% EtOH (3×20 L) at room temperature. The EtOH extracts were evaporated under reduced pressure to afford a dark brown semi-solid (250 g), which was then suspended in water and partitioned sequentially with petroleum ether (30 g), dichloromethane (18 g), and EtOAc (6.5 g). A portion of the EtOAc fraction (1.06 g) was subjected to silica gel column chromatography and was eluted with a gradient of petroleum ether and EtOAc (2:1 to 1:4) to yield three fractions (Fr. [1] [2] [3] . The most active fraction was then eluted with a gradient the mixture of CH 2 Cl 2 and MeOH (from 2:1 to 1:2) to afford another three fractions (Fr. [4] [5] [6] , of which Fr. 5 was further purified by reversed-phase silica gel (RP-18) with an eluent of CH 3 CN : MeOH: H 2 O (4 : 4 : 2) to afford PPa (25 mg) . Dark green solids, 1 The cytotoxicity of PPa on MDCK cells was evaluated by MTT assay as described before 17 . Briefly, MDCK cells were prepared in 96-well plates (1 x 10 4 cells for each well) for 24 h and exposed to PPa in a 2-fold serial dilution. After incubation for 48 h, 100 μL of MTT (Sigma, USA) solution (0.5 mg/mL DMEM diluent) was added and left at 37 °C for 4 h. Subsequently, the supernatant was removed, and 150 μL of DMSO solution was added to plate to dissolve the formazan product. The absorbance of each well was measured at 570 nm by using a Multiskan FC microplate reader (Thermo Fisher Scientific, Massachusetts, USA). MDCK cells (ATCC) were cultured in 96-wellg plates (2 × 10 4 cells/well) for 24 h. A series of double-diluted PPa solutions was pre-incubated with 100 TCID 50 of the virus at 37 °C for 30 min, and these cells were incubated with a virus-compound mixture for 1 h after two washes with PBS. Then, 1 μg/mL of TPCK-trypsin (trypsin treated with TPCK, Sigma, USA) in serum-free DMEM was added to the cells. Next, cell viability was measured using the MTT method at 48 h after the infection. S-KKWK 17 was used as a positive control, and the experiment was independently repeated at least three times. Virus subtypes such as the A/FM-1/1/47 (H1N1) mouse-adapted strain, A/Puerto Rico/8/34 (H1N1), A/Puerto Rico/8/34 (H1N1) with the NA-H274Y mutation, and A/Aichi/2/68 (H3N2) were selected to evaluate the antiviral effects of PPa. Anti-SARS-CoV-2 assay was processed as reported previously 18 . Briefly, 5×10 4 Vero-E6 cells/well were seeded in 48-well plates at 37℃ overnight. To start the assay, SARS-CoV-2 virus (MOI of 0.05) was pre-incubated with gradiently diluted PPa at 37℃ for 30 min, then the mixture was transferred to the cells and incubated for another 1 h. After incubation, cells were washed with PBS and added with the fresh medium for 24 h. Then cell supernatants were collected and subjected to viral RNA isolation, then qRT-PCR was performed to measure expression of the S gene of SARS-CoV-2 18 . Generally, serially diluted PPa in 100 μL 2% FBS DMEM were incubated with 200 plaque forming units (PFUs) of RSV (A2 strain) in equal volume of 2% FBS DMEM for 1 hour at 37°C. Then 150 μL mixture were added to each well of 24-well plate seeded with Vero-E6 cell to near confluency. Then the plate was incubated at 37°C for 1 hour before replacing the mixture with 1% methylcellulose DMEM. Five days later, methylcellulose medium was thoroughly removed and cells were fixed with 4% formaldehyde, permeabilized with 0.1% triton and blocked with 5% nonfat-milk. Mouse antisera against RSV Fusion protein diluted at 1:300 w incubated with cells for 1 hour at 37°C and washed three time by PBS-0.1% Tween 20. Cells were then incubated with second antibody-conjugated with horseradish peroxidase (catalogue number: SA00001, Proteintech, China) for 1 hour at room temperature and washed three times as usual. Finally, plaques appeared 15 mins later after adding 3, 3', 5, 5'-tetramethylbenzidine reagent. Plaques in each well were manually counted, no-compound well were made as control in each test. Four different drug administration protocols were used in this study to assess the possible mechanisms of PPa, as described in a previous study 19 MDCK cells were cultured in 6-well plates (4×10 5 cells/well) for 24 h. Antiviral effects were evaluated using two drug treatment approaches: "During infection" and "Pretreatment of the virus", as mentioned above 17 . After treatment, cells were washed with PBS to remove the free virus and then cultured in 3 mL of serum-free MEM (2×) containing 1× TPCK-trypsin (1.6% AGAR for 72 h, as mentioned above). The cells were then fixed with 4% paraformaldehyde for 20 min, and then incubated with a 0.5% (w/v) crystal violet dye solution for 1 h at 37 °C. The effect of PPa on viral plaque formation was determined by counting the number of plaques. MDCK cells cultured in 6-well plates (4 x 10 5 cells for each well) were subjected to two modes of drug treatment: "during infection" and "pretreatment virus". After 24 h of post-infection, total cellular RNA was extracted with Trizol reagent (Sigma, St. Louis, MO, USA) and reverse transcribed into cDNA using the primers as listed below. Real-time quantitative PCR was performed using the two-step PCR amplification standard procedure of ABI7500 system (Applied Biosystems, Massachusetts, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Sigma, USA) was used as an internal control. The relative expression of the HA gene was measured by a classical 2 −ΔΔCT method by using 7500 software. Each sample was tested independently more than three times. The primer sequences of target genes are (GAPDH-Reverse). To study which phase of the virus life cycle PPA interacted with, the infected MDCK cells were treated with 5 μg/mL of PPa for the indicated time interval (0−2, 2−4, 4−6, 6−8 h), covering the first viral replication cycle. After 24 h, the cells were frozen and thawed three times until the cells were completely disrupted, the supernatant was collected, the cell debris was removed by centrifugation, and the virus titer was determined by a method to measuring TCID 50 of virus. The polykaryon formation inhibition assay was performed using a protocol described to 800 nm. The effects of the PPa interaction with lipids were further investigated by measuring the fluorescence intensity. Briefly, lipids were suspended in PBS at a concentration of 1 mg/mL and incubated with 1 μg/mL of PPa in the dark for 2 h. The fluorescence emission spectrum was detected using a fluorescence spectrophotometer and compared with the sample lacking lipids. Lipids extracted from MDCK cells were dissolved in 5% DMSO at a concentration of Subsequently, the mice were orally administered different agents once a day for five days. On the sixth day, the mice were provided access to the food and water ad libitum. Body weights, mortality and the general behavior of the mice were recorded daily for 15 consecutive days. Using the same protocol as the in vivo anti-influenza virus test described above, the intragastric administration was stopped in three mice from each group on the fourth day after infection with the virus and the animals were sacrificed. Their lungs were harvested, washed with normal saline, dried with gauze and weighed. The lung index was calculated using the following equation: Lung index = lung weight / body weight × 100 % After evaluating the lung index, all of the lung tissues obtained from mice were immersed in 4% paraformaldehyde solution (PFA, LEAGENE) as soon as possible. Then the lung tissues were embedded in paraffin, cut into thin sections (about 4 μm thick), and stained using the Hematoxylin and Eosin staining method before histopathologic analysis under the microscope. Statistics were performed using GraphPad Prism 5 software (San Diego, CA). Data expressed as the means ± standard deviation (SD) were repeated at least three times. ImageJ was used to quantized the Fluorescent images. Data were determined by one-way ANOVA using SPSS 22.0 software. The mortality rates were analyzed by Log-rank (Mantel-Cox) test (P < 0.01) using GraphPad Prism 5 software. Statistical significance was defined as * P < 0.05, ** P < 0.01, *** P < 0.001. The mussel M. senhousei was collected in the area of the South China Sea, Guangdong Province, China. The fresh mussels were grounded and extracted with ethanol at least three times. The crude extracts were then isolated and purified using a propanoic acid, also named pyropheophorbide a (PPa) 15 , as shown in Fig. 1a . < Fig. 1 > We then tested the antiviral activity of In addition, the MTT assay was performed to evaluate the cytotoxicity of the tested molecules and to determine whether the anti-IAV activity resulted from the cytotoxicity of PPa. As shown in Table 1 , the CC 50 value of PPa was 74.84±1.93 μg/mL, a value that is much higher than its viral inhibitory activity. The anti-IAV activity of PPa was next confirmed using the plaque reduction assay. As indicated in Fig. 1b and 1c . 2a) and then quantified using ImageJ software 21 (Fig. 2b) . The green fluorescence associated with NP expression was significantly decreased compared with the "virus" group without drug treatment. Notably, the "Pretreatment" group showed greater activity of the drug than the simultaneous treatment group ("During infection"). A similar conclusion was obtained by measuring the mRNA level of the HA gene from influenza A/PR/8/34 (H1N1) after treatment with PPa using two drug administration approaches (Fig. 2c) . The level of HA gene was dramatically decreased when treated with 5 µg/mL PPa compared with the group without drug treatment (P<0.001), and the HA level observed in cells subjected to the "Pretreatment of the virus" was lower than in cells subjected to the "During infection" treatment. Furthermore, the antiviral activity of PPa was also assessed by testing viruses that induce respiratory disease, including respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As indicted in Fig. 2d , at 25 µM, PPa inhibited the formation of RSV plaques up to 75% detected by immuno-based plaque assay, and inhibited almost 100% of the expression of S gene of SARS-CoV-2 detected by qRT-PCR (Fig. 2d) . Therefore, in addition to influenza A viruses, PPa was also active against other viruses that cause respiratory diseases. Next, we investigated the inhibitory effects of PPa on the influenza virus life cycle. Four different drug administration approaches, including Pretreatment of cells, Pretreatment of the virus, During infection, and After infection, were employed as described above 19 to test the inhibitory effects of the drug. The CPEs were observed under a microscope and used to evaluate the antiviral effects of PPa (Fig. 3a) , which were further quantified using the MTT assay. As shown in Table 2 In addition to HA, we also investigated if the surface glycoprotein neuraminidase (NA) was the possible target of PPa. As shown in Fig. 3e , PPa did not noticeably inhibit NA inhibition at the concentrations tested. In the NA inhibition assay performed with a concentration of PPa as high as 32 µg/mL, the percent inhibition of the activity of NA toward the substrate 4-MU-NANA was still less than 25%. Since the viral envelope lipid bilayer was derived from the host cell membrane, we next investigated the interactions between PPa and lipids that were extracted from MDCK cells. First, we tested whether PPa bound to the surface of the lipid bilayer. In this experiment, 5 μg/mL PPa was incubated with lipid extracts or with MDCK cells at 37°C for 45 min. After extensive rinses with PBS to remove the unbound PPa, the PPa absorbed on lipid or cells was then extracted with methanol, and the fluorescence intensity was subsequently measured at the excitation wavelength of 365 nm. As shown in Fig. 4c , an intense fluorescence emission of PPa at a wavelength of ca. 666 nm was observed from lipid extracts (60% remaining) and cell extracts (38% remaining), indicating a strong binding affinity of PPa for the lipid bilayer. Next, fluorescence spectroscopy was employed to measure the interactions between PPa and lipids. First, 1 µg/mL of PPa in PBS or mixed with 1 mg/mL of lipids in PBS was excited at a wavelength of 365 nm, and then the emission fluorescence was measured at wavelengths ranging from 640 to 720 nm, as indicated in Fig. 4d . The data revealed a ca. 15-fold increase in the fluorescence intensity when the assay was performed in the presence of lipids. Furthermore, an apparent red shift of 11 nm from 666 nm to 677 nm in the maximum emission wavelength was observed when lipids were added to PPa. Inspired by these data, we then semi-quantitatively measured the lipid-PPa interactions using isothermal titration calorimetry (ITC) to obtain the thermodynamic curve of the binding of PPa to lipids 24 . The lipids extracted from MDCK cells were added at a concentration 1 mg/mL, while the concentration of PPa was 75 μg/mL. The thermal changes due to the interactions between PPa and cellular lipids were detected and presented in the thermogram shown in Fig. 4e . The ITC peaks were negative, indicating that the interactions between PPa and lipids were exothermic. Based on the measured ΔH and TΔS values ( Because PPa interacts with the lipid bilayer, we next examined the effect of lipids on the anti-IAV activity of PPa in vitro. As indicated in Table 4 , the antiviral activity of PPa was apparently reduced after the addition of lipids, further supporting the presence of interactions between PPa and lipids. The anti-IAV activity of PPa was further assessed by analyzing IAV-infected mice. In the experiment, 4-week-old male Kunming mice (the average body weight was ca. 19-22 g) were divided into five groups of 10 mice each, as indicated in Fig. 5a As shown in Fig. 5a , compared with the virus control group, the survival rate and mean survival time of infected mice treated with both high and low dosages of PPa were noticeably increased. In parallel, a significant increase in the lung index was observed for all virus-challenged mice compared with the blank control group. In addition, the average lung indices for compound-treated groups were lower than the virus control group; nevertheless, no significant difference between PPa-H group and PPa-L group was observed in this experiment (Fig. 5b) . Furthermore, the body weight of all virus-infected mice decreased until the eighth to ninth days compared with the slow increase in body weight observed in the blank control group. The body weights of compound-treated groups slowly increased, while all the mice in virus control group had died (Fig. 5c) . In addition, the histopathological examination of the lungs of mice (Fig. 5d) showed that as compared with the blank control group, a large amount of alveolar collapse or deformation around bronchioles occurred, and many inflammatory cell infiltrations were observed in the virus-infected mice. In contrast, the symptoms were dramatically alleviated in the compound-treated group (Fig. 5d) , further supporting the protective effect of PPa. humans and affects 5-15% of the global population with a mild to severe illness each year 25 . Currently, only two types of anti-IAV drugs are available, but the emergence of viral strains resistant to these compounds has been reported 26 , in addition to some side effects. Therefore, it is imperative to develop new and effective anti-IAV drugs that employ a different mechanism by targeting other viral proteins or cellular factors involved in the influenza virus life cycle. In this study, we identified a porphyrin derivative, pyropheophorbide a (PPa), with broad anti-IAV activity in vitro. PPa was isolated from the marine mussel M. senhousei, and it has also been isolated from abalone 27 . Structurally, PPa is a derivative of chlorophyll. Therefore, a reasonable deduction that the original sources of PPa might be phytoplankton, which were subsequently transformed by mussels to produce PPa 28 . As shown in Fig. 1a , the structure of PPa consists of a porphyrin ring and a carboxyl group, which shows a typical amphipathic structure. This characteristic facilitates PPa to interact with hydrophobic lipids of enveloped virions or cell membrane, by which to block the entry of virus 29 . When the carboxyl group connected with a lipid chain as that in chlorophyll, which decreased the amphipathicity of PPa, and as a result, decreased the antiviral activity of PPa. However, more and extensive structure-activity relationship studies should be conducted to support this observation. In the present study, the anti-IAV activity of PPa was assessed using various experiments, including CPE, RT-PCR, a plaque reduction assay, and indirect immunofluorescence staining. These data, In contrast to those reported viral entry inhibitors, our data showed that although PPa and its derivatives functioned as virus entry inhibitors and exhibited strong binding affinities for hemagglutinin (HA) and HA2 subunit in an experiment using surface plasmon resonance (SPR) (data not shown), the negative results of the hemagglutinin inhibition assay and hemolysis inhibition assay suggested that its major target may not be HA, as a consequence, PPa and its derivatives were unable to block In conclusion, in this paper, we identified a porphyrin derivative of PPa from the marine mussel M. senhousei that shows broad anti-IAV activity in vitro and in vivo, as well as other viral strains that cause respiratory diseases including RSV and SARS-CoV-2. PPa interacted with the viral lipid bilayer to alter membrane-associated functions, thereby inhibiting the entry of enveloped viruses. However, additional, more extensive experiments are still needed to further assess the antiviral efficacy, the detailed mechanism by which PPa inhibits virus entry into the host cells, and particularly, the in vivo anti-IAV effects of PPa. Nevertheless, as we have known, porphyrins are a group of conjugated planar molecules that possess broad applications in the field of biomedical science, due to their unique structural features and electrochemical performance 39 . Our findings in this study expand the application of porphyrins, and significantly, by targeting an essential component in the virus life cycle and using PPa or its derivatives as a leading compound, we believe that a more potent antiviral agent may be generated. Table 3 Thermodynamic parameters of the interaction between PPa and lipids from MDCK cells Table 4 Anti-Influenza virus inhibition rate after PPa interacted with lipids from MDCK cells Afterwards, 4-MU-NANA was added to each well, the mixture was incubated for another 1 h, and the reaction was terminated with NaOH (83% ethanol). Finally, an excitation wavelength of 340 nm and an emission wavelength of 440 nm were used to measure resulting fluorescence of the mixture. Oseltamivir phosphate and zanamivir were employed as positive controls. The mortality rates were analyzed with Pearson's Chi-square test (P < 0.05). 5b Lung index of infected mice treated with PPa. The lung index of the mice was determined after the viral infection and PPa treatment for 3 days. 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