key: cord-1047828-kxtmf174
authors: Siniavin, Andrei E.; Nikiforova, Maria A.; Grinkina, Svetlana D.; Gushchin, Vladimir A.; Starkov, Vladislav G.; Osipov, Alexey V.; Tsetlin, Victor I.; Utkin, Yuri N.
title: Snake venom phospholipases A2 possess a strong virucidal activity against SARS-CoV-2 in vitro and block the cell fusion mediated by spike glycoprotein interaction with the ACE2 receptor
date: 2021-01-12
journal: bioRxiv
DOI: 10.1101/2021.01.12.426042
sha: 0cbae8dd49f08f6cd9d40b9b31ed8d3d65070c4b
doc_id: 1047828
cord_uid: kxtmf174

A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the absence of specific therapeutic and prophylactic agents, the virus has infected almost hundred million people, of whom nearly two million have died from the viral disease COVID-19. The ongoing COVID-19 pandemic is a global threat requiring new therapeutic strategies. Among them, antiviral studies based on natural molecules are a promising approach. The superfamily of phospholipases A2 (PLA2s) consists of a large number of members that catalyze the hydrolysis of phospholipids at a specific position. Here we show that secreted PLA2s from the venom of various snakes protect to varying degrees the Vero E6 cells widely used for the replication of viruses with evident cytopathic action, from SARS-CoV-2 infection PLA2s showed low cytotoxicity to Vero E6 cells and the high antiviral activity against SARS-CoV-2 with IC50 values ranged from 0.06 to 7.71 μg/ml. Dimeric PLA2 HDP-2 from the viper Vipera nikolskii, as well as its catalytic and inhibitory subunits, had potent virucidal (neutralizing) activity against SARS-CoV-2. Inactivation of the enzymatic activity of the catalytic subunit of dimeric PLA2 led to a significant decrease in antiviral activity. In addition, dimeric PLA2 inhibited cell-cell fusion mediated by SARS-CoV-2 spike glycoprotein. These results suggest that snake PLA2s, in particular dimeric ones, are promising candidates for the development of antiviral drugs that target lipid bilayers of the viral envelope and may be good tools to study the interaction of viruses with host cell membranes.

The running title: Snake phospholipases show virucidal activity to SARS-CoV-2 1 0 1 1

In December 2019, a rapidly spreading community-acquired pneumonia was discovered in Wuhan (Hubei 4 1 Province, China) and subsequent studies have shown that this disease is caused by a virus belonging to the 4 2 coronavirus family, this conclusion soon confirmed by sequencing the full-length genome from samples 4 3 (bronchoalveolar lavage fluid) of patients with pneumonia 1 . In February 2020, WHO 4 4 (https://www.who.int/dg/speeches/detail/who-director-general-s-remarks-at-the-media-briefing-on-2019-4 5 ncov-on-11-february-2020) has officially named this disease «COVID-19» (coronavirus disease 2019) and 4 6

Next, we studied the antiviral activity of two isolated subunits of HDP-2. The results revealed striking 1 3 1 differences between their actions: the catalytically active subunit HDP-2P showed 2-fold higher antiviral 1 3 2 activity than the parent HDP-2, while enzymatically inactive subunit HDP-1I showed two orders of 1 3 3 magnitude lower antiviral activity than HDP-2 (Fig. 2 ). This suggests that antiviral activity may be related 1 3 4 to phospholipolytic activity. To determine the contribution of the phospholipase activity of the HDP-2P 1 3 5 subunit to the inhibition of SARS-CoV-2, the enzymatic activity of this subunit was inhibited by chemical 1 3 6 modification of the active site (HDP-2P inact). It was found that after such modification the antiviral 1 3 7 activity of HDP-2P decreased 70-fold ( Fig. 1 and Fig. 2 ). Thus, PLA2s from different snakes showed 1 3 8 various antiviral activity against SARS-CoV-2. The abolishment of enzymatic activity resulted in a strong 1 3 9 decrease of antiviral activity. To evaluate the direct effects of PLA2s on the Vero E6 cells, the cytotoxicity of PLA2 was studied: it was 1 4 2 found that they slightly slow down cell proliferation, without morphological changes in the cell monolayer 1 4 3 (Fig. 3) . The highest, albeit moderate, cytotoxicity was manifested by Vur-PL2 and HDP-2P which at 100 1 4 4 μ g/ml reduced cell proliferation on average by 38% and 51%, respectively. Only HDP-2P at the maximal 1 4 5 concentration has pronounced cytotoxicity with a change in cell morphology. However, HDP-2P inhibited 1 4 6 SARS-CoV-2 CPE at concentration of 0.1 µg/ml, that is at three order of magnitude lower concentration. subunits, a significant suppression of the infectious viral titer was achieved (Fig. 4) . Complete suppression 1 5 7 of the infectivity of SARS-CoV-2 was observed when the viral stock was treated with the catalytically 1 5 8 active subunit HDP-2P at all tested concentrations. However, as in the experiment on antiviral activity, 1 5 9

inhibition of the enzymatic activity of HDP-2P led to a loss in the ability to completely inactivate SARS-1 6 0

CoV-2, which was manifested by an increase in viral titer at lower concentration of HDP-1I or modified 1 6 1 HDP-2P. 1 6 2

To study cell fusion mediated by S-glycoprotein interaction with the ACE2 receptor, we used 293T cells 1 6 4 expressing green fluorescent protein (GFP) and SARS-CoV-2 S-glycoprotein (293T-GFP-Spike) as well 1 6 5 as Vero E6 cells expressing ACE2 which is a receptor for glycoprotein S. The effect of the five snake 1 6 6 venom PLA2s on the SARS-CoV-2 S-glycoprotein mediated cell-cell fusion was investigated. After co-1 6 7 cultivation of effector 293T-GFP-Spike cells and target Vero E6 cells at 37 0 C for 2 h in the presence of 1 6 8 various concentrations of PLA2s, the number of fused cells, having the size at least 2 times larger than 1 6 9 normal cells and multiple nuclei, were counted using a fluorescence microscope. It was found that at 1 7 0 8 concentrations from 1 to 100 µg/ml the dimeric HDP-1 and HDP-2 inhibited S-glycoprotein mediated 1 7 1 cell-cell fusion. They showed approximately the same concentration-dependent activity inhibiting cell-cell 1 7 2 fusion by 70% at 100 µg/ml (Fig. 5) . At concentration of 1 µg/ml, this value decreased to about 48%, still 1 7 3 manifesting statistically significant difference from control. Vur-PL2 and BF-PLA2-II at a concentration 1 7 4 of 100 µg/ml showed insignificant inhibition, while HDP-2P at 100 µg/ml completely blocked the cell-cell In the present study, we have demonstrated that snake sPLA2s have antiviral activity against SARS-CoV-1 8 7 2. The highest activity was exhibited by dimeric PLA2s of the group IIA. Strong virucidal activity against 1 8 8 SARS-CoV-2 was observed when viral particles were treated with HDP-2 or its subunits, especially with 1 8 9 enzymatically active subunit HDP-2P. However, the virucidal and antiviral activity of the HDP-2P was 1 9 0 markedly suppressed when the His residue in the active center was modified by 4-bromophenacyl 1 9 1 bromide, a specific inhibitor of PLA2s. It is interesting to note that a previous study of PLA2 CM-II 1 9 2 3 4 they block the gp120-CD4 interactions, which are important for the initiation of conformational changes 2 3 5

in the viral envelope that trigger virus entry into the host cells 80 . To check the homology between 2 3 6 glycoprotein S and dimeric PLA2s, we have performed the alignment of the glycoprotein S amino acid 2 3 7 sequence with those of HDP-2 and HDP-1. Some similarity between the sequences was observed (Fig. 6) .

Interestingly, the amino acid sequences of PLA2s have similarity to the glycoprotein S fragment 413-462, 2 3 9 which according to the X-ray structure is involved in interaction with ACE2 81 . Amino acid residues 2 4 0 Lys417, Tyr449 and Tyr453 forming contacts with ACE2 are conserved in PLA2 sequences. Considering 2 4 1 this homology, we suggest that dimeric PLA2s may compete with SARS-CoV-2 for binding to ACE2.

However, a true mechanism for blocking S-glycoprotein mediated cell-cell fusion by PLA2s remains to be 2 4 3 studied.

In this study, we have demonstrated for the first time the antiviral activity of snake PLA2s against SARS-2 4 5

CoV-2. Dimeric HDP-2 and its catalytic subunit showed high virucidal activity, most likely due to the 2 4 6 cleavage of glycerophospholipids on the virus envelope, which can lead to the destruction of the lipid 2 4 7 bilayer and destabilization of surface glycoproteins in the virus. Summarizing previous and our findings 2 4 8 on the ability of snake PLA2s to inactivate various viruses, these results highlight the potential of PLA2s 2 4 9 as a natural product for the development of broad-spectrum antiviral drugs. Phospholipase A2 II (BF-PLA2-II, GenBank AAK62361.1) and phospholipase A2 1 (BF-PLA2-1, 2 6 4 UniProtKB Q90WA7) were purified from krait Bungarus fasciatus venom as described 71 . Phospholipase 2 6 5 A2 Vur-PL2 (UniProtKB F8QN53) was purified from viper V. ursinii renardi venom as described in 82 .

Dimeric phospholipases HDP-1 and HDP-2 were isolated from vipera V. nikolskii venom and separated 2 6 7 into subunits HDP-1P (UniProtKB Q1RP79), HDP-2P (UniProtKB Q1RP78) and HDP-1I (UniProtKB 2 6 8 A4VBF0) as described in 36 .

Modification of HDP-2P 2 7 0 4-Bromophenacyl bromide (Lancaster, England) as 10 mM stock in acetone was added to final 2 7 1 concentration of 200 µM to the 20 µM HDP-2P solution in 50 mM Tris HCl buffer, pH 7.5, containing 10 2 7 2 mM Na 2 SO 4 . The mixture was incubated for 6 h at a room temperature and separated using a Jupiter C18 2 7 3 HPLC column (Phenomenex) and acetonitrile gradient from 20 to 50% in 30 min in the presence of 0.1% 2 7 4

Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus 4

Spike Protein by the Transmembrane Protease TMPRSS2

A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus 4