key: cord-0684518-xuwgxus6
authors: Kudryashova, Elena; Zani, Ashley; Vilmen, Geraldine; Sharma, Amit; Lu, Wuyuan; Yount, Jacob S.; Kudryashov, Dmitri S.
title: Inhibition of SARS-CoV-2 infection by human defensin HNP1 and retrocyclin RC-101
date: 2021-09-03
journal: J Mol Biol
DOI: 10.1016/j.jmb.2021.167225
sha: 7b4406c167881f98929ce457ea7b18d034eadbc6
doc_id: 684518
cord_uid: xuwgxus6

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 pandemic. The emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that can inhibit the virus. One component of the innate immune system with broad antipathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The ability of defensins to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of proteins with high conformational plasticity. We found that human neutrophil α-defensin HNP1 binds to SARS-CoV-2 Spike protein with submicromolar affinity that is more than 20 fold stronger than its binding to serum albumin. As such, HNP1, as well as a θ-defensin retrocyclin RC-101, both interfere with Spike-mediated membrane fusion, Spike-pseudotyped lentivirus infection, and authentic SARS-CoV-2 infection in cell culture. These effects correlate with the abilities of the defensins to destabilize and precipitate Spike protein and inhibit the interaction of Spike with the ACE2 receptor. Serum reduces the anti-SARS-CoV-2 activity of HNP1, though at high concentrations, HNP1 was able to inactivate the virus even in the presence of serum. Overall, our results suggest that defensins can negatively affect the native conformation of SARS-CoV-2 Spike, and that α- and θ-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.

COVID-19 imposes an extraordinary health threat whose scale and severity can be compared only to that of the Spanish flu pandemic, which raged over a hundred years ago. The COVID-19 pandemic vividly exposed the weakness of the globalized world to new zoonotically transmitted viruses, which emerge with alarming regularity (e.g., 2003 Severe Acute Respiratory Syndrome coronavirus SARS-CoV [1] , 2009 influenza A H1N1 [2] , 2012 Middle East Respiratory Syndrome coronavirus MERS-CoV [3] ). Although several effective SARS-CoV-2 vaccines have been generated in an unprecedently short time frame, high levels of transmissibility and mortality of this virus (reviewed in [4] ), hesitancy towards vaccination from a large fraction of the world population, and the emergence of variants resistant to vaccine-induced immunity require additional development of non-vaccine therapies against SARS-CoV-2.

Interestingly, despite the high mortality rate, as much as 42% of infected individuals are asymptomatic carriers of SARS-CoV-2 [5] [6] [7] , suggesting that the virus can be effectively controlled by the human innate immune system [8] . Recently, pro-and antiviral activities of interferoninduced transmembrane proteins (IFITMs) in SARS-CoV-2 infection have been described [9] [10] [11] .

Likewise, other interferon-stimulated genes, such as CH25H, Ly6E, Tetherin, and ZAP have been demonstrated to have inhibitory effects on SARS-CoV-2 replication in vitro [12] [13] [14] [15] [16] . Another important and promising class of innate immunity effectors is defensins. These peptides are active against several viruses, including human immunodeficiency virus (HIV), herpes simplex virus, influenza virus, and SARS-CoV [17] [18] [19] [20] .

Based on their structural characteristics, mammalian defensins are grouped into three subfamilies called α-, β-, and θ-defensins [21] [22] [23] [24] [25] . Humans express α-and β-defensins, while cyclic θ-defensins are produced only by non-human primates [26] . Although human θ-defensin genes are transcribed, a premature stop codon precludes their translation [27] . Intriguingly, human cells retain the ability to produce the cyclic θ-defensin peptides upon transfection of the synthetic "humanized" θ-defensin genes called retrocyclins [28] . Potent antiviral and antibacterial activity [26, [29] [30] [31] in combination with low cytotoxicity and exceptional stability identified retrocyclins as promising topical microbicides [32] [33] [34] .

α-Defensin human neutrophil peptides 1-4 (HNP1-4) are small (~30 a.a.) dimeric cationic amphiphilic peptides produced by neutrophils and stored in azurophil granules [35] . Highly similar HNP1-3 peptides differ from each other only by their first amino acid residue and constitute >98% of the total neutrophil α-defensins [36] . HNP1 shows potent antibacterial [37] [38] [39] [40] , antifungal [41, 42] , and antiviral activities against enveloped and non-enveloped viruses (reviewed in [17, 18, 43] ). Antiviral mechanisms of defensins (reviewed in [44] ) are multifaceted, including direct targeting of exposed proteins on viral envelopes and capsids, disruption of viral fusion, inhibition of post-entry processes, or affecting receptors on the host cell surface. Such remarkable versatility is partially explained by the ability of defensins to disrupt membranes [39] , but also by their ability to induce unfolding of metastable proteins, such as bacterial toxins and viral proteins [45] [46] [47] . Susceptibility of target proteins to α-defensin-induced unfolding and ultimately inactivation is reliant on their conformational plasticity, which often correlates with thermolability (reviewed in [48] ). Near the melting point, proteins exist in equilibrium between folded and partially unfolded states. Unlike the majority of ligands, which bind to folded conformations of protein targets and increase their termostability, defensins promote unfolding of thermolabile proteins, reducing their melting temperatures [47] . Defensin targets become locked in non-native, partially unfolded, and therefore, nonfunctional states and cannot undergo necessary conformational transitions and often aggregate/precipitate through exposed hydrophobic surfaces. As evolutionary derivatives of α-defensins [26] , θ-defensins (and retrocyclins, in particular) mainly recapitulate their activity [49] . Defensins are also recognized as chemokines that modulate the immune response by binding to cell surface receptors and interfering with cellular signaling [44] .

According to a recent study, enteric human α-defensin HD5 binds to human angiotensinconverting enzyme 2 (a.k.a. ACE2 receptor) on enterocytes obstructing the SARS-CoV-2 Spike binding site, which results in inhibition of SARS-CoV-2 Spike pseudovirus infection [50] .

Encouraged by this report, we examined the effects of a neutrophil α-defensin, HNP1, and retrocyclin, RC-101, on SARS-CoV-2 infection. We chose to focus on HNP1, since it is the most relevant α-defensin for the respiratory virus infection, as it is produced by neutrophils and is released from their granules at the sites of infection/inflammation. RC-101 replicates most of the α-defensin functions and is a promising candidate for drug development.

SARS-CoV-2 homotrimeric Spike glycoprotein binds to the ACE2 receptor on the host cell, mediating membrane fusion and virus entry [51] . Being exposed on the surface of virions, Spike is the main target for virus neutralization by host antibodies. Membrane fusion activity of Spike also enables fusion of Spike-expressing mammalian cells with ACE2-positive cells to form multinuclear cell syncytia [52] . Such syncytia represent a convenient model to study Spike/ACE2 interaction and to test different agents in disrupting this interaction. An additional approach that also does not require biosafety level 3 (BSL3) conditions is to use replication-restricted viruses pseudotyped with SARS-CoV-2 Spike. We initially utilized both systems to test whether HNP1 and RC-101 can interfere with the SARS-CoV-2 Spike interaction with host cells. Since serum has been shown to negatively affect antipathogen activity of defensins [40, 53, 54] , we tested their effects in the presence and absence of serum.

Co-transfection of human osteosarcoma U2OS cells with Spike and mCherry promoted their fusion with ACE2-producing human lung Calu-3 cells, leading to multinuclear mCherry-positive syncytia (Fig. 1A,B) . In the absence of serum, the addition of HNP1 or RC-101 to the co-culture of Spike-and ACE2-positive cells strongly inhibited syncytia formation (Fig. 1B,C) , indicating that defensins are able to inhibit Spike-mediated membrane fusion. Serum largely abrogated the inhibition of syncytia formation by HNP1 (Fig. 1D) , suggesting that abundant serum proteins may compete for the defensin binding.

In the next set of experiments, human lung H1299 cells were infected with HIV-based reporter virus-like particles pseudotyped with SARS-CoV-2 Spike ( Fig. 2A) ; pseudovirus infection was monitored using a secreted NanoLuc luciferase (Nluc) reporter encoded by the virus genome.

Pre-treatment of the cells with HNP1 or RC-101 prior to the infection did not affect the pseudovirus infection efficiency (Fig. 2B,C) , demonstrating that these defensins did not provide a cell-directed effect on infection. In contrast, one-hour pre-incubation of the pseudovirus particles with the defensins prior to cell infection significantly inhibited the infection (Fig. 2D) . These results suggest that HNP1 and RC-101 can interfere with the viral infection by acting upon the viral particle rather than influencing host molecules (e.g., ACE2 receptor), as it has been suggested for the enteric defensin HD5 [50] . As with the Spike-mediated fusion experiments, serum had a negative influence on the inhibitory ability of HNP1 and RC-101 (Fig. 2E ).

The results from the SARS-CoV-2 Spike-mediated membrane fusion assays and pseudovirus infections prompted us to test whether HNP1 can directly interact with Spike protein.

Fluorescence anisotropy revealed that Cy5-HNP1 strongly binds to recombinant SARS-CoV-2 Spike with submicromolar affinity (K d = 146  18 nM; Fig. 3A ). Given the inhibitory effect of serum and the recognized role of serum proteins as defensin scavengers [55] , we measured whether Cy5-HNP1 can also bind serum albumin, the most abundant and highly interactive serum protein.

Indeed, Cy5-HNP1 was able to bind to bovine serum albumin (BSA), but with over 20-fold lower affinity (K d = 3.4  0.3 M; Fig. 3B ) as compared to Spike, indicating a much higher selectivity in binding the viral protein.

Defensins inactivate various structurally unrelated pathogen effector proteins such as bacterial toxins and viral proteins [45, 47, 49] . A common trait that unites these targets is their marginal thermodynamic stability dictated by the need for transitioning through dramatic conformational perturbations required for passing through narrow pores (i.e., for bacterial toxins) or fusing viral and host membranes (i.e., viral fusion proteins) [45, 47, 56] . For viral proteins, conformational plasticity is also dictated by an ability of such proteins to tolerate high mutational loads, often needed for evading adaptive immune responses. SARS-CoV-2 Spike is subject to these evolutionary pressures and, as such, is anticipated to be prone to inactivation by defensins.

Therefore, we assessed the effects of HNP1 and RC-101 on the stability of Spike using differential scanning fluorimetry (DSF) [57] . Spike protein was destabilized at micromolar concentrations of HNP1 and RC-101 (Fig. 3C ,D) as implied from the impaired melting profile with a loss of the melting peak at its normal position and a higher signal at low temperatures ( Fig. 3C ). Such behavior is characteristic of chemical denaturation [47] as it reports the fluorophore binding to hydrophobic residues of Spike exposed at temperatures below the original melting point of thermal denaturation. Since the signs of Spike destabilization by RC101 were less prominent than for HNP1, we sought for additional experimental evidence confirming the mechanism of the inhibition.

Binding of HNP1 and RC-101 and locking of unfolded protein conformations can result in protein aggregation through exposed hydrophobic surfaces. Indeed, partial aggregation and precipitation of Spike was observed by pelleting upon incubation with HNP1 or RC-101 (Fig. 3E ).

These results imply that the dominant conformational state of Spike is compromised in the presence of defensins, which, therefore, are likely to interfere with its functional activity. Yet, given relatively mild destabilization and precipitation effects of RC101, additional or alternative mechanisms of Spike inhibition by defensins cannot be completely ruled out.

To test whether defensins can alter the Spike/ACE2 interaction, we developed a solid-phase binding assay (Fig. 3F ). We opted for surface attachment of biotinylated ACE2 on a NeutrAvidincoated plate followed by blocking the plate with BSA. In this case, SARS-CoV-2 Spike could be pre-treated with defensins in the absence of BSA prior to addition to ACE2-bound plate. To detect Spike protein bound to ACE2 we used anti-Spike S2 subunit antibody. In the absence of either ACE2 or Spike, the background signal was low, validating specificity of the assay (Fig. 3G ). Higher background noise was detected in the presence of defensins at concentrations above 5 µM, which limited our ability to test the effects of higher doses of the peptides. Pre-incubation of Spike in the presence of HNP1 or RC-101 for 1 h at 37 o C inhibited Spike/ACE2 binding in a concentrationdependent manner (Fig. 3G ). Together our results demonstrate that defensins can bind SARS-CoV-2 Spike, alter its conformation, and impair its interaction with ACE2.

We next evaluated whether the inhibitory effects of HNP1 and RC-101 observed in the pseudovirus infections also occur when studying genuine SARS-CoV-2 infections in a BSL3- strongly inhibited the SARS-CoV-2 infection even in the presence of 10% serum, providing that the peptide was present at sufficiently high concentration (e.g., 43 µM; Fig. 5B ,C).

The data presented in this manuscript reveal that SARS-CoV-2 infection can be inhibited by two distinct defensins, and identify Spike as the target responsible for the observed inhibition.

Defensins bound Spike with submicromolar affinities, promoted its unfolding and precipitation, and negatively affected interaction of Spike with ACE2, the receptor for SARS-CoV-2 entry into human cells. The quantitative difference in the efficiency of the defensin effects on SARS-CoV-2 vs pseudotyped lentiviral infection (notably more potent for authentic virus) in our study may be explained by several variables that differ in the experiments: the nature of parent viruses (SARS-CoV-2 vs HIV), cells (Vero E6 vs H1299), reporters (immunodetection of Spike followed by flow cytometry vs a luminescent reporter), and the level of serum (less controlled in the case of lentiviral assay due to technical reasons). The direct comparison of the K d of HNP1 to Spike with the IC 50 upon virus inhibition in cellular assays is similarly impractical due to several factors: the unknown stoichiometry of the inhibition, differences in solution and surface biochemistry, and the presence of other highly abundant binding partners of HNP1 (e.g., cell and virus membranes, to mention few) in the cellular assays.

While this manuscript was under revision, a complementary study was published that corroborates our main findings that α-and θ-defensins inhibit SARS-CoV-2 infection [59] . In agreement with our results, Xu and coauthors [59] found that HNP1 blocked Spike-mediated viral fusion; the inhibitory effects of defensins were more potent upon pre-incubation with the virus, in agreement with our conclusion that the inhibition is mediated by targeting viral rather than host molecules. These authors also attempted to assess the effects of defensins on Spike/ACE2 interactions using a solid-phase binding approach but failed to observe any inhibition, which may be due to a suboptimal experimental design. They chose to pre-treat ACE2 with defensins before its addition to immobilized Spike receptor-binding domain (RBD). Since ACE2 is an unlikely target (Figs. 4, 5) , but is higher than those required to inhibit the interaction between Spike and ACE2 in the solid-phase assay (Fig. 3G) . This difference may reflect a distribution of defensins between additional targets present in cellular assays (e.g., membranes, other proteins).

Alternatively, higher doses of defensins may be required to overcome the avidity of the highly multimeric cell-virus interaction. Of note, since the recombinant Spike protein used in our in vitro assays was stabilized in the prefusion conformation by mutations (K986P and V987P), the destabilizing effects of defensins on native Spike might be even more prominent.

Although destabilization of Spike by defensins, resulting in its compromised interaction with ACE2, is a likely mechanism behind the inhibition of SARS-CoV-2 infection of human cells, we recognize that the effects of defensins on viruses are multifaceted [18] and may extend beyond the inhibition of Spike/ACE2 interaction reported here. Due to the broad nature of defensin interactions with affected proteins, other mechanisms (e.g., blocking protease cleavage, or preventing conformational changes needed for fusion) may contribute to the inhibition of Spike by defensins. Although destabilization, as reflected in lower melting temperatures, is the extreme manifestation of the defensins on target proteins, lower doses of defensins likely cause lessnotable perturbations to the protein structure, which can, nevertheless, be functionally consequential. Most importantly, the pliable, metastable nature of viral proteins is a primary reason for their higher susceptibility to the effects of defensins compared to most host proteins.

The ability of defensins to get integrated into the hydrophobic core of such proteins defines the unusual combination of a broad specificity and high affinity of these interactions.

In our study, the ability of α-, and θ-defensins to promote unfolding of pathogenic proteins [45] is confirmed on a yet another viral pathogen. We propose that this ability plays a key role in various aspects of the protective mechanisms employed by defensins when viral proteins are involved. Perhaps counterintuitively, even the reported stabilization of viral capsids by defensins adenovirus is about 220 ng/mL (~0.07 µM) [61] . In peripheral blood of COVID-19 patients, the levels of HNP1-3 vary from ~1.5 to 7.6 nM [62] . However, these low levels can be misleading given that HNP1-4 defensins are produced by neutrophils, whose effects are typically restricted to local environments where they are most needed. Indeed, higher local levels of HNP1-4 than those needed for inhibition of SARS-CoV-2 are expected near neutrophils before dilution by body fluids [63] . Furthermore, the highly interactive, "sticky" nature of defensins and their ability to cause precipitation of partner proteins is another factor contributing to higher local and lower systemic levels of defensins.

Therapeutic application of defensins and other antimicrobial peptides as antiviral agents has been suggested [64, 65] . Since the inhibitory effects of the HNP1 and RC-101 against SARS-CoV-2 are diminished by serum, the defensins perhaps can be most effective as topical antiviral agents (e.g., intranasal). Similarly, retrocyclins have been proposed as topical microbicides to prevent sexually transmitted infections caused by HIV-1 [66, 67] . Notably, owing to their cyclic nature, high stability, and resistance to proteolysis, retrocyclins are particularly promising as therapeutic agents. Retrocyclins have low toxicity in cell cultures and in vivo, are well tolerated in animal models, and are non-immunogenic in chimpanzees [68, 69] . Together our data suggest that HNP1 and RC-101 should be further evaluated as candidates for developing topical anti-COVID-19 and broad-range antiviral drugs.

HNP1 and RC-101 were prepared by solid-phase peptide synthesis, and the correct folding was ensured as described previously [70] [71] [72] [73] . The activity of HNP1 and RC-101 was confirmed using the recombinant actin cross-linking domain (ACD) of Vibrio cholerae MARTX toxin [47] . 

All cells were cultured at 37°C with 5% CO 2 in a humidified incubator. U2OS and Vero E6 cells were grown in Dulbecco's Modified Eagle Medium, H1299 -in RPMI-1640 medium, Calu-3 -in Eagle's Minimum Essential Medium, all supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). H1299, Calu-3, Vero E6, and HEK293T cells were purchased from ATCC. The identity and purity of the U2OS cells were verified by STR profiling (Amelogenin + 9 loci) at the Genomic Shared Resource (OSU, Comprehensive Cancer Center) with 100% match using The Cellosaurus cell line database [75] . All cell lines were mycoplasma-negative as determined by a PCR-based approach [76] . 

DSF was performed as described previously [47, 56] . Briefly, SARS-CoV-2 Spike protein was diluted to 1 M (calculated as monomer concentration) in PBS in the absence or presence of HNP1 or RC-101 and supplemented with 1:5000 dilution of Sypro Orange dye (Invitrogen, Carlsbad, CA). Changes in fluorescence of the dye, which preferentially binds to protein hydrophobic regions exposed upon thermal-induced unfolding, were measured using a CFX realtime PCR detection system (Bio-Rad, Hercules, CA). The melting temperatures (T m ) were determined as the maximum of the first derivative (dF/dT) of each normalized experimental curve.

To assess protein precipitation, 1 M of Spike protein (calculated as monomer concentration; Additional controls included Spike-treated wells with and without pre-incubation with defensins in the absence of ACE2.

Virus-like particles pseudotyped with SARS-CoV-2 Spike protein were produced by transfecting where I max is the maximal inhibition, D is the concentration of defensin, n is the Hill coefficient.

Data are presented as mean values from several independent experiments (as indicated in the 

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AUTHORS CONTRIBUTIONS EK: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing -Original Draft, Writing -Review & Editing, Visualization AZ: Investigation, Writing -Review & Editing GV: Investigation, Writing -Review & Editing

Funding acquisition WL: Resources, Writing -Review & Editing JSY: Formal analysis, Resources, Writing -Review & Editing, Supervision, Project administration, Funding acquisition DSK: Conceptualization, Methodology, Validation, Resources, Writing -Original Draft, Writing -Review & Editing, Supervision

 Human defensin HNP1 binds to SARS-CoV-2 Spike protein with submicromolar affinity 

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: