Article Inhibit SARS-CoV-2 Infection by Blocking Viral Entry

Chuan Xu 1,†, Annie Wang 1,†, Mariana Marin 2, William Honnen 1, Santhamani Ramasamy 1, Edith Porter 3 , Selvakumar Subbian 1 , Abraham Pinter 1 , Gregory B. Melikyan 2 , Wuyuan Lu 4,* and Theresa L. Chang 1,5,*

1 Public Health Research Institute, Rutgers, New Jersey Medical School, The State University of New Jersey, Newark, NJ 07103, USA; [email protected] (C.X.); [email protected] (A.W.); [email protected] (W.H.); [email protected] (S.R.); [email protected] (S.S.); [email protected] (A.P.) 2 Department of Pediatrics, Infectious Diseases Emory University, Atlanta, GA 30322, USA; [email protected] (M.M.); [email protected] (G.B.M.) 3 Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032, USA; [email protected] 4 Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Science, and Shanghai Institute of Infectious Disease and Biosecurity, Fudan University, Shanghai 200032, China 5 Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers, New Jersey Medical School, The State University of New Jersey, Newark, NJ 07103, USA * Correspondence: [email protected] (W.L.); [email protected] (T.L.C.) † These authors contributed equally.

Abstract: Innate immunity during acute infection plays a critical role in the disease severity of   severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and is likely to contribute to COVID-19 disease outcomes. Defensins are highly abundant innate immune Citation: Xu, C.; Wang, A.; Marin, factors in and epithelial cells, including intestinal Paneth cells, and exhibit antimicrobial M.; Honnen, W.; Ramasamy, S.; and immune-modulatory activities. In this study, we investigated the effects of human α- and β- Porter, E.; Subbian, S.; Pinter, A.; defensins and RC101, a θ- analog, on SARS-CoV-2 infection. We found that human Melikyan, G.B.; Lu, W.; et al. Human (HNPs) 1–3, human defensin (HD) 5 and RC101 exhibited potent antiviral activity against Defensins Inhibit SARS-CoV-2 Infection by Blocking Viral Entry. pseudotyped viruses expressing SARS-CoV-2 spike . HNP4 and HD6 had weak anti-SARS- Viruses 2021, 13, 1246. https:// CoV-2 activity, whereas human β-defensins (HBD2, HBD5 and HBD6) had no effect. HNP1, HD5 doi.org/10.3390/v13071246 and RC101 also inhibited infection by replication-competent SARS-CoV-2 viruses and SARS-CoV-2 variants. Pretreatment of cells with HNP1, HD5 or RC101 provided some protection against viral Academic Editor: Daniel R. Perez infection. These defensins did not have an effect when provided post-infection, indicating their effect was directed towards viral entry. Indeed, HNP1 inhibited viral fusion but not the binding of the Received: 24 May 2021 spike receptor-binding domain to hACE2. The anti-SARS-CoV-2 effect of defensins was influenced Accepted: 22 June 2021 by the structure of the peptides, as linear unstructured forms of HNP1 and HD5 lost their antiviral Published: 26 June 2021 function. Pro-HD5, the precursor of HD5, did not block infection by SARS-CoV-2. High titers overcame the effect of low levels of HNP1, indicating that defensins act on the virion. HNP1, HD5 Publisher’s Note: MDPI stays neutral and RC101 also blocked viral infection of intestinal and epithelial cells. The protective effects of with regard to jurisdictional claims in defensins reported here suggest that they may be useful additives to the antivirus arsenal and should published maps and institutional affil- be thoroughly studied. iations.

Keywords: SARS-CoV-2; defensins;

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative conditions of the Creative Commons agent of coronavirus disease 19 (COVID-19), has spread worldwide with more than 179 mil- Attribution (CC BY) license (https:// lion cases and 3.8 million deaths as of late-June 2021 [1]. The ability of the innate immune creativecommons.org/licenses/by/ response to limit the magnitude of virus replication during acute infection is known to 4.0/). determine the severity of disease outcomes of coronaviruses [2–4]. Identification of mucosal

Viruses 2021, 13, 1246. https://doi.org/10.3390/v13071246 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 1246 2 of 15

factors against SARS-CoV-2 is critical for developing antiviral treatments and therapeutic options for COVID-19. Defensins, antimicrobial peptides that play a crucial role in innate immunity, are abundant in neutrophils and are expressed by epithelial cells at mucosal sites [5–7]. Neutrophils have been shown to infiltrate the lung in response to SARS-CoV-2 infection in animal models [8–10], suggesting their function in the control of virus replica- tion, although dysregulation of neutrophils may contribute to hyperinflammation in severe COVID-19 diseases [11]. Human defensins are cationic peptides with β-pleated sheet structures that are sta- bilized by three intramolecular disulfide bonds between residues [5–7]. These defensins are classified into the α-, β- and θ-defensins based on the disulfide links be- tween conserved cysteine residues and their structure [5–7]. The effects of defensins on viral infection are defensin-, virus- and cell-type-specific [12–14]. Human α-defensins including human neutrophil peptides (HNPs) 1–4 and human α-defensins 5 and 6 (HD5 and HD6), which are mainly produced by neutrophils and intestinal Paneth cells, re- spectively, exhibit antiviral activities against enveloped and non-enveloped viruses [12]; however, evidence indicates that HD5 and HD6 can also promote viral infectivity of HIV and some adenoviruses [15,16]. Human β-defensins (HBDs) do not appear to affect virions, but these peptides are known to suppress viral replication by altering cellular functions [14]. Theta-defensins (θ-defensins), which are cyclic octadecapeptides expressed in rhesus macaques and baboons, and retrocyclins (RC), which are synthesized based on human θ-defensin , also exhibit antiviral activities [17,18]. In addition to their effects on virus replication, defensins serve as immune modulators to regulate innate and adaptive immunity [19,20]. For example, defensins exhibit inflammatory and anti-inflammatory activities, and recruit and activate T cells and myeloid cells such as monocytes and dendritic cells [19,20]. Administration of rhesus θ-defensin-1 (RTD-1) pre- vents death in MA15-SARS-CoV-infected mice by reducing pulmonary pathology. The mechanism is thought to involve immune modulation, as RTD-1 does not exhibit anti- SARS-CoV activity in vivo or in vitro [21]. Here, we have determined the effects of various human defensins and a θ-defensin analog RC101 on SARS-CoV-2 infection in vitro and their underlying mechanisms.

2. Materials and Methods 2.1. Reagents, Plasmids, and Cell Lines Defensins and their analogs were chemically synthesized, folded and verified as described previously [22]. To construct linear unstructured analogs of HNP1 and HD5, [Abu]HNP1 and [Abu]HD5, the six cysteine residues were replaced by isosteric α-aminobu tyric acid (Abu). Recombinant HD5 propeptides (aa 20-94) were synthesized using a baculovirus/insect cell culture system [23]. Recombinant SARS-CoV-2 spike receptor- binding domain (RBD) and hACE2 proteins, expressed in HEK293T cells, were purchased from RayBiotech (Peachtree Corners, GA, USA). The CCF4-AM β-lactamase substrate (GeneBLAzer in vivo detection kit) was purchased from Invitrogen (Carlsbad, CA, USA). The construct for full-length SARS-CoV-2-Wuhan-Hu-1 surface (spike) (GenBank accession number QHD43416) [24] was codon-optimized for and synthesized with Kozak-START GCCACC ATG and STOP codons and with 50 Nhel/30Apal sites for subcloning into the pcDNA3.1(+) vector (Thermo Fisher Scientific, Waltham, MA, USA). Plasmids encoding spike proteins of B.1.1.7 and P1 variants were kindly provided by Dennis Burton (The Scripps Research Institute, La Jolla, CA, USA). The packaging HIV-1 pR9∆Env (from Chris Aiken at Vanderbilt University), pMM310 expressing β-lactamase-Vpr chimera (BlaM-Vpr, provided by Michael Miller at Merck Research Laboratories and now available from NIH AIDS Research and Reference Program), pMDG-VSV-G plasmid expressing VSV-G (from J. Young at Roche Applied Science, Mannheim, Germany), psPAX2 lentiviral packaging vector and pcRev vector have been described previously [25]. pCAGGS-SARS- CoV-2 S D614G (cat.#156421) and pWPI-IRES-Puro-Ak-ACE2-TMPRSS2 (cat. #154987) expression vectors were obtained from Addgene (Watertown, MA, USA). Viruses 2021, 13, 1246 3 of 15

HEK293T/17, Vero E6, Caco-2 and A549 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). HEK293T-hACE2 cells and HeLa-hACE2 cells were kindly provided by Hyeryun Choe (The Scripps Research Institute, Jupiter, FL, USA) [26] and by Dennis Burton [27], respectively. Huh7.5 was obtained from Dr. Charles Rice (Rockefeller University) through Apath LLC (Brooklyn, NY, USA). To generate Huh7.5- ACE2-TMPRSS2 cells for viral fusion assay, Huh7.5 cells grown at 60% confluency in six-well plates were transduced with single-cycle infectious VSV-G pseudotyped lentiviral viruses (0.5 ng p24/well) encoding for ACE2-TMPRSS2 by centrifugation at 16 ◦C for 30 min at 1550× g. After 24 h transduction, cells were transferred in a 10-cm tissue culture dish in the presence of 2 µg/mL puromycin.

2.2. Cell Culture HEK 293T, HEK293T-hACE2, HeLa-hACE2, Caco-2 and A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum. For HEK293T/17 cells, the growth medium was supplemented with 0.5 mg/mL of G418 sulfate (Mediatech, Inc). Huh7.5-hACE2-TMPRSS2 cells were cultured in complete media with 2 µg/mL puromycin.

2.3. Production of Pseudotyped Viruses Replication-defective HIV-1 luciferase-expressing reporter viruses pseudotyped with SARS-CoV-2 S proteins were produced by co-transfection of a plasmid encoding the envelope-deficient HIV-1 NL4-3 virus with the luciferase reporter gene (pNL4-3.Luc.R-E- or pNL4-3.Luc.R+E- from N. Landau, New York University) and a pcDNA3.1 plasmid expressing the SARS-CoV-2 glycoprotein into HEK 293T cells, which were seeded at 6–7 × 106 in a 10-cm dish and cultured overnight, using Lipofectamine 3000 (Thermo Fisher Scientific) as described previously [28]. The supernatant was collected 48 h after transfection and filtered. Virus stocks were analyzed for HIV-1 p24 antigen by the AlphaLISA HIV p24 kit (PerkinElmer). Because serum is known to reduce the effect of defensins on the virion [29,30], viruses were produced under serum-free conditions, or for viruses generated in 10% FBS, these were diluted in serum-free medium (to a final FBS concentration of 3%). To produce serum-free SARS-CoV-2 pseudotyped viruses, the culture media were replaced by DMEM without serum at 24 h after transfection, and the cells cultured for an additional 24 h prior to collecting viruses. Virus stocks contained approximately 200 ng/mL of HIV p24 proteins. For infection assays, cells plated at 2 × 104 or 5 × 104 cells/well in a 96-well or 48-well plate, respectively, were cultured overnight. Pseudotyped SARS-CoV-2 luciferase reporter viruses were incubated with defensins at 37 ◦C for 1 h. The defensin-virus mixture was then added to cells. After 1–2 h viral attachment, infected cells were cultured in media with 10% FBS for 48–72 h before measuring luciferase activity using Luciferase Substrate Buffer (Promega Inc). Luciferase activity (relative light units; RLU.) reflecting viral infection was measured on a 2300 EnSpire Multilabel Plate Reader (PerkinElmer, Waltham, MA). Results obtained using different titers of viruses are reported as the average percentage of the control calculated using the formula: (RLU of treated cells / RLUs of untreated cells) × 100. To generate BlaM-Vpr harboring pseudotyped SARS-CoV-2 viruses for the viral fusion assay, HEK293T/17 cells grown at 75% confluency in a 10-cm dish were transfected with pCAGGS-SARS-CoV-2 S D614G (4 µg), pR9∆Env (4 µg), BlaM-Vpr (2 µg) and pcRev (0.5 µg) using the JetPRIME transfection reagent (Polyplus-transfection, Illkirch-Graffenstaden, France). Transfected cells were incubated for 14h at 37 ◦C and then cultured in a fresh growth medium for an additional 36 h. The viral supernatants were filtered through 0.45 µm polyethersulfone filters (VWR, Radnor, PA, USA) and concentrated 5x using Lenti-X™ Concentrator (Clontech, Mountain View, CA, USA). To produce the transducing VSV-G pseudotyped viruses for generating Huh7.5-hACE2-TMPRSS2 cells, HEK293T/17 cells were transfected with pMDG-VSV-G (1.5 µg), psPAX2 (3 µg) and ACE2-TMPRSS2 expression vectors (4 µg). Viruses were prepared as described above. Viruses 2021, 13, 1246 4 of 15

2.4. Replication-Competent Virus Infection Replication-competent SARS-CoV-2 viruses expressing mNeonGreen, kindly provided by Pei-Yong Shi at the University of Texas Medical Branch, Galveston, TX, USA, were propagated in Vero E6 cells as described previously [31]. Experiments were performed in a biosafety level 3 laboratory with personal protection equipment including powered air-purifying respirators (Breathe Easy, 3M), Tyvek suits, aprons, sleeves, booties and double gloves. Virus titers were determined by plaque assays in Vero E6 cells as described previously [32]. For the infection assay, Vero E6 cells at 1.5 × 104 cells/well in a black 96-well glass plate (Greiner, Monroe, NC, USA) were incubated overnight. Cells were exposed to viruses (30 µL) with or without defensin treatment at a multiplicity of infection (MOI) of five for 1 h followed by the addition of 100 µL FluoroBrite medium containing 2% FBS. Fluorescence from productive viral infections was monitored at 48 h after infection using a Biotek Cytation 5 multi-mode plate reader.

2.5. Viral Attachment Assay HeLa-hACE2 cells seeded at 5 × 104 per well in 48-well plates were cultured overnight. Pseudotyped viruses were pretreated with or without defensins at 37 ◦C for 1 h. Cells were incubated with viruses at 4 ◦C for 2 h, washed with cold PBS three times, and lysed with 100 µL of 1% Triton X-100. Cell-associated HIV p24 was determined by AlphaLISA HIV p24 kit (PerkinElmer).

2.6. SARS-CoV-2 RBD and hACE Binding Assay Greiner Microlon 200 plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with recombinant SARS-CoV-2 RBD proteins at 50 ng/well in 0.1 M bicarbonate buffer pH 9.6 at 4 ◦C for 24 h. The plates were blocked with 2% bovine serum albumin in PBS (w/v) for 30 min at 37 ◦C, washed with PBS, and incubated with biotinylated hACE2 pro- teins (0.1 µg/mL) that were pre-treated with defensins at 37 ◦C for 1 h. After washing, the bound hACE2 proteins were detected by incubating with -conjugated streptavidin at 37 ◦C for 1 h. Plates were washed and the binding of hACE2 proteins to SARS-CoV-2 RBD was detected by adding 1 mg/mL of ρ-nitrophenol in DEA buffer at pH 9.8, and measuring the resulting signal at 405 nm.

2.7. Virus-Cell Fusion Assay The virus-cell fusion assay was performed as described previously [33]. Briefly, pseu- dotyped SARS-CoV-2 viruses containing a β-lactamase-Vpr chimera (BlaM-Vpr) were diluted in the medium without FBS (0.5 ng p24/well), bound to target cells by centrifuga- tion at 16 ◦C for 30 min at 1550× g. Unbound viruses were removed and a growth medium containing or lacking defensins was added. Virus-cell fusion was initiated by incubating the samples at 37 ◦C for 2 h. Cells were then loaded with the CCF4-AM fluorescent substrate and incubated overnight at 11 ◦C. The cytoplasmic BlaM activity (ratio of blue to green fluorescence) was measured using the SpectraMaxi3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA).

2.8. Cytotoxicity Assay HEK 293T-hACE2 cells were plated in 96-well plates at 5000 cells per well and then treated with various concentrations of defensins for 24 h. Cell viability was analyzed using MTS-based CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA).

2.9. Statistical Analysis Statistical comparisons were performed using a two-tailed Independent-Samples t- test; p < 0.05 was considered significant. Prism 8 (GraphPad Software, LLC, San Diego, CA, USA) was used for these analyses. Viruses 2021, 13, x FOR PEER REVIEW 5 of 15

MTS‐based CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Mad‐ ison, WI, USA).

2.9. Statistical Analysis Statistical comparisons were performed using a two‐tailed Independent‐Samples t‐ Viruses 2021, 13, 1246 test; p < 0.05 was considered significant. Prism 8 (GraphPad Software, LLC, San Diego,5 of 15 CA, USA) was used for these analyses.

3. Results 3.3.1. Results Human α‐Defensins Inhibit SARS‐CoV‐2 Infection 3.1. Human α-Defensins Inhibit SARS-CoV-2 Infection To assess the effect of defensins on SARS‐CoV‐2 infection, pseudotyped luciferase virusTo particles assess the expressing effect of defensins SARS‐CoV on‐ SARS-CoV-22 surface (spike, infection, S) proteins pseudotyped were luciferasetreated with virus or particleswithout expressingdefensins for SARS-CoV-2 1 h before surface infection (spike, of HEK293T S) proteins‐hACE2 were cells, treated as withdescribed or without in the defensinsMethods. for Viral 1 h infection before infection was determined of HEK293T-hACE2 by measuring cells, the as luciferase described activity in the Methods.in cells at Viralday three infection post‐ wasinfection. determined Note that by measuringdefensins were the luciferasenot added activity back after in attachment. cells at day HNPs three post-infection.1–3 blocked SARS Note‐CoV that defensins‐2 infection, were achieving not added approximately back after attachment. 50% suppression HNPs 1–3 at blocked1 μg/mL SARS-CoV-2 infection, achieving approximately 50% suppression at 1 µg/mL (290 nM) (290 nM) (Figure 1A–C). Because HNPs 1–3 differ by only a single (N‐terminal) amino (Figure1A–C). Because HNPs 1–3 differ by only a single (N-terminal) residue, it is acid residue, it is not surprising that all three peptides had similar effects. HNP4, a low‐ not surprising that all three peptides had similar effects. HNP4, a low-abundance in abundance peptide in neutrophils and less known for its antiviral activity, was substan‐ neutrophils and less known for its antiviral activity, was substantially less potent (Figure1D ). tially less potent (Figure 1D). Two intestinal defensins (HD5 and HD6) were also studied. Two intestinal defensins (HD5 and HD6) were also studied. HD5 exhibited significant HD5 exhibited significant anti‐SARS‐CoV‐2 activity, achieving 60% suppression at 12.5 anti-SARS-CoV-2 activity, achieving 60% suppression at 12.5 µg/mL (3.45 µM), whereas μg/mL (3.45 μM), whereas HD6 only blocked SARS‐CoV‐2 infection at the highest con‐ HD6 only blocked SARS-CoV-2 infection at the highest concentration tested (50 µg/mL, centration tested (50 μg/mL, 13 μM) (Figure 1E,F). The antiviral activity of defensins was 13 µM) (Figure1E,F) . The antiviral activity of defensins was not associated with any observed not associated with any observed cytotoxicity (Supplemental Figure S1). Of note, the con‐ cytotoxicity (Supplemental Figure S1). Of note, the concentrations of HNPs 1–3 and HD5 centrations of HNPs 1–3 and HD5 employed in these experiments were within the physi‐ employed in these experiments were within the physiological ranges of these defensins in neutrophilsological ranges and inof intestinalthese defensins tissues in and neutrophils lumen [5, 34and]. in intestinal tissues and lumen [5,34].

ABC HNP3 2000020 HNP1 2000020 HNP2 2000020

1500015 15000 1500015

15 3 3 3 * * 1000010 1000010 * 1000010 RLUs x10 RLUs x10 * * RLUs x10 * * 50005 50005 * 50005 * * * 0 0 0 0 1 12.5 25 50 g/ml 0 1 12.5 25 g/ml 0 1 12.5 25 50 g/ml

D E F 40000 HNP4 HD5 HD6 4 400004 600006

300003 300003 4 4 400004 4 200002 200002 * * * * RLUs x10

RLUs x10 2

20000RLUs x10 100001 100001 *

0 0 0 0 1 12.5 25 50 g/ml 0 1 12.5 25 50 g/ml 0 1 12.5 25 50 g/ml

Figure 1. Human α-defensins inhibit infection by pseudotyped SARS-CoV-2 virus. Pseudotyped luciferase reporter virus Figure 1. Human α‐defensins inhibit infection by pseudotyped SARS‐CoV‐2 virus. Pseudotyped luciferase reporter virus expressing SARS-CoV-2 S was incubated with or without HNPs1-4, HD5 or HD6 at 37 ◦C for 1 h. Viruses with expressing SARS‐CoV‐2 S protein was incubated with or without HNPs1‐4, HD5 or HD6 at 37 °C for 1 h. Viruses with or or without defensin treatment were used to infect HEK293T cells expressing hACE2 (A–F) as described in the Methods. Infected cells were cultured for three days before measuring luciferase activity. The significance of the differences between defensin-treated virions and mocked-treated controls was calculated by Student’s two-tailed, unpaired t = test; * p < 0.05.

Data are means ± SD and are representative of four independent experiments.

Unlike human α-defensins, β-defensins HBD2, HBD5 and HBD6 did not effectively inhibit viral infection (Figure2A–C). However, the θ-defensin analog RC101 exhibited a similar level of inhibition of SARS-CoV-2 infection (50% inhibition at 6.4 µg/mL, 3.4 µM) Viruses 2021, 13, x FOR PEER REVIEW 6 of 15

without defensin treatment were used to infect HEK293T cells expressing hACE2 (A–F) as described in the Methods. Infected cells were cultured for three days before measuring luciferase activity. The significance of the differences between defensin‐treated virions and mocked‐treated controls was calculated by Student’s two‐tailed, unpaired t = test; *p < 0.05. Data are means ± SD and are representative of four independent experiments.

Viruses 2021, 13, 1246 Unlike human α‐defensins, β‐defensins HBD2, HBD5 and HBD6 did not effectively6 of 15 inhibit viral infection (Figure 2A–C). However, the θ‐defensin analog RC101 exhibited a similar level of inhibition of SARS‐CoV‐2 infection (50% inhibition at 6.4 μg/mL, 3.4 μM) as HNPs 1–3 and HD5 (Figure 2D). The human α‐defensins as well as RC101 largely main‐ as HNPs 1–3 and HD5 (Figure2D). The human α-defensins as well as RC101 largely tained their antiviral activities in the presence of serum (Figure 2E,F). maintained their antiviral activities in the presence of serum (Figure2E,F).

A B C 400004 HBD2800008 HBD5 800008 HBD6

300003 600006 600006 4 4 4

200002 400004 400004 RLUs x10 RLUs x10 RLUs x10 100001 200002 200002

0 0 0 0 1 12.5 25 50 g/ml 00.55 50100g/ml 00.55 50100g/ml

D E F RC101 800008 RC101 1200008 800008 virus with 10% FBS Virus with 10% FBS

600006 900006 600006 4 4 4 * * 400004 600004 400004 * * * RLUs x10 RLUs x10 * RLUs x10 * * * 200002 300002 * 200002 * * 0 0 0 0 1 12.5 25 50 g/ml 0 1 12.5 25 50 g/ml 3 6 P1 P2 D trol N N HD5 H H H HNP con 20 g/ml 50 g/ml

FigureFigure 2. 2. ThetaTheta defensin defensin analog analog RC101 RC101 but but not not HBDs HBDs inhibits inhibits pseudotyped pseudotyped SARS SARS-CoV-2‐CoV‐2 infection. infection. Pseudotyped Pseudotyped luciferase luciferase reporterreporter viruses viruses expressing expressing SARS SARS-CoV-2‐CoV‐2 S S proteins proteins were were incubated incubated with with or or without without the the indicated indicated concentrations concentrations of of HBD2, HBD2, ◦ HBD5,HBD5, HBD6 HBD6 or RC101 at 3737 °CC forfor 11 hh followedfollowed by by infection infection of of HEK293T HEK293T cells cells expressing expressing hACE2 hACE2 (A (–AD–)D as) as described described in thein theMethods. Methods. Infected Infected cells cells were were cultured cultured for for three three days days before before measuring measuring luciferase luciferase activity. activity. The The effect effect of defensinsof defensins on theon thevirus virus in the in the presence presence of 10%of 10% FBS FBS was was also also determined determined (E ,(FE). and Differences F). Differences between between defensin-treated defensin‐treated virions virions and non-treated and non‐ treatedcontrols controls were calculated were calculated by Student’s by Student’s two-tailed, two unpaired‐tailed, unpairedt-test; * p t<‐test; 0.05. * Datap < 0.05. are meansData are± SDmeans and ± are SD representative and are repre of‐ sentativethree independent of three independent experiments. experiments.

WeWe then then determined determined whether whether defensins defensins blocked blocked replication replication-competent‐competent SARS SARS-CoV-2‐CoV‐2 viruses.viruses. Replication Replication-competent‐competent SARS-CoV-2 SARS‐CoV‐2 viruses viruses expressing expressing mNeonGreen mNeonGreen were were incubated incu‐ batedwith HNP1,with HNP1, HD5 andHD5 RC101 and RC101 at different at different concentrations concentrations for 1 h for before 1 h infectionbefore infection of Vero of E6 Verocells. E6 In cells. agreement In agreement with results with from results pseudotyped from pseudotyped SARS-CoV-2 SARS viruses‐CoV‐2 (Figure viruses1), (Figure HNP1, 1),HD5 HNP1, and HD5 RC101 and suppressed RC101 suppressed viral infection viral in infection a dose-dependent in a dose‐dependent manner (Figure manner3). (Figure 3). SARS-CoV-2 variants, including P.1 and B.1.1.7 from Brazil and the United Kingdom, respectively, are highly transmissible and increase the risk of death [35–38]. We determined the effect of defensins on pseudotyped viruses expressing spike proteins from the P.1 and B.1.1.7 variants. HNP1, HD5 and RC101 suppressed the P.1 variant in a dose-dependent manner (Figure4A). HNP1, HD5 and RC101 exhibited moderate antiviral activity against the B.1.1.7 variant (Figure4B). HNP1 and HD5 at 50 µg/mL, which inhibited the earlier Wuhan strain by 96% (Figure1), suppressed P1 infection by 67% and 72%, respectively, suggesting that the emerged variant was more resistant to host antimicrobial peptides. Sim- ilarly, HNP1 and HD5 at 50 µg/mL inhibited B.1.1.7 infection by 58% and 32%, respectively. Although RC101 suppressed infection by both variants, it was also less potent against the B.1.1.7 variant (Figure4). Viruses 2021, 13, x FOR PEER REVIEW 7 of 15

A B C HNP1 RC101 HD5 3 3 80003 8 80008 80008

60006 60006 60006 *

40004 * 40004 40004 * * * * 20002 * 20002 20002

Fluorescent Intensity Fluorescent Intensity Fluorescent * * * 0 0 0 Fluorescence Intensity x10 Fluorescence Intensity x10 Fluorescence Intensity x10 0 1 5 25 50 g/ml 0152550g/ml 0152550g/ml

Figure 3. DefensinsViruses inhibit2021, 13infection, x FOR PEER REVIEW by replication‐competent SARS‐CoV‐2 viruses. Replication‐competent SARS‐CoV‐2 7 of 15 Viruses 2021, 13, 1246 7 of 15 viruses expressing mNeonGreen (at MOI of five) were incubated with HNP1 (A), HD5 (B) and RC101 (C) at different concentrations and at 37 °C for 1 h before addition to Vero E6 cells for 2 h. Infected cells were then cultured in FluoroBrite media with 10% FBS for two days. The fluorescence from productive viral infections was measured using Biotek Cytation A B C 5. The differences between defensin‐treated virions and non‐treated controls were calculated by Student’s two‐tailed, un‐ HNP1 RC101 HD5 3 3 paired t‐test; * p < 0.05. Data are8000 3 means8 ± SD and are representative80008 of three independent experiments.80008

60006 60006 60006 SARS‐CoV‐2 variants, including P.1 and* B.1.1.7 from Brazil and the United Kingdom,

respectively,40004 are* highly transmissible40004 and increase the risk40004 of death [35–38]. We deter‐ * * mined the effect of defensins on pseudotyped viruses* expressing spike proteins* from the P.1 2000and2 B.1.1.7 variants.* HNP1,2000 HD52 and RC101 suppressed20002 the P.1 variant in a dose‐

Fluorescent Intensity Fluorescent Intensity Fluorescent * * * dependent0 manner (Figure 4A). HNP1,0 HD5 and RC101 exhibited0 moderate antiviral ac‐ Fluorescence Intensity x10 Fluorescence Intensity x10 Fluorescence Intensity x10 tivity against0 1 the 5 B.1.1.7 25 50 variantg/ml (Figure0152550 4B). HNP1 andg/ml HD5 at 500152550 μg/mL, whichg/ml inhibited Figure 3.theDefensins earlier inhibit Wuhan infection strain by by replication-competent 96% (Figure 1), SARS-CoV-2 suppressed viruses. P1 infection Replication-competent by 67% and SARS-CoV-2 72%, re‐ virusesFigurespectively, expressing3. Defensins mNeonGreen inhibitsuggesting infection (at that MOI by replication the of five) emerged were‐competent incubated variant SARS with was‐CoV HNP1 ‐more2 viruses. (A ),resistant HD5 Replication (B) andto ‐hostcompetent RC101 antimicrobial (C) SARS at different‐CoV‐2 concentrationsvirusespeptides. expressing and at Similarly,mNeonGreen 37 ◦C for 1 hHNP1 before(at MOI additionand of five) HD5 to were Vero at incubated E650 μ cellsg/mL for with 2 h.inhibited HNP1 Infected (A cells), B.1.1.7 HD5 were (B then)infection and cultured RC101 by ( inC ) FluoroBrite 58%at different and mediaconcentrations with 10% and FBS forat 37 two °C days. for 1 h The before fluorescence addition fromto Vero productive E6 cells for viral 2 h. infections Infected was cells measured were then using cultured Biotek in CytationFluoroBrite 5. media 32%,with 10% respectively. FBS for two days. Although The fluorescence RC101 fromsuppressed productive infection viral infections by both was measuredvariants, using it was Biotek also Cytation less The differences between defensin-treated virions and non-treated controls were calculated by Student’s two-tailed, unpaired 5. The potentdifferences against between the defensin B.1.1.7‐treated variant virions (Figure and non 4).‐treated controls were calculated by Student’s two‐tailed, un‐ t-test; * p < 0.05. Data are means ± SD and are representative of three independent experiments. paired t‐test; * p < 0.05. Data are means ± SD and are representative of three independent experiments.

A P.1 variant (Brazil) SARS‐CoV‐2 variants, including P.1 and B.1.1.7 from Brazil and the United Kingdom,

7 HNP1respectively, are highly transmissibleHD5 and increase7 the riskRC101 of death [35–38]. We deter‐ 2.5×102.5 2.5×102.57 2.5×102.5 mined the effect of defensins on pseudotyped viruses expressing spike proteins from the 2.0×102.07 P.1 and B.1.1.72.0×10 variants.2.07 HNP1, HD5 and 2.0×10RC1012.07 suppressed the P.1 variant in a dose‐ 7 7 7 * 7 * 1.5×101.57 dependent manner1.5×101.5 (Figure7 4A).* HNP1, HD51.5×10 and1.5 RC101 exhibited moderate antiviral ac‐ tivity against the B.1.1.7 variant (Figure 4B). HNP1 and HD5 at 50 μg/mL, which inhibited 7 1.0×101.07 1.0×101.07 1.0×101.0 RLUs x10 RLUs x10 RLUs x10 * the earlier Wuhan strain by 96% (Figure* 1), suppressed P1 infection by 67% and 72%, re‐ 5.0×100.56 spectively, suggesting5.0×100.56 that the emerged variant5.0×100.5 was6 more resistant to host antimicrobial peptides. Similarly, HNP1 and HD5 at 50 μg/mL inhibited B.1.1.7* infection by 58% and 0.00 0.00 0.00 0105032%, respectively.g/ml Although01050 RC101 suppressedg/ml infection01050 by both variants,g/ml it was also less potent against the B.1.1.7 variant (Figure 4). B B.1.1.7 variant (UK) A P.1 variant (Brazil) 4000004 HNP14000004 HD54000004 RC101 7 HNP1 HD5 7 RC101 2.5×102.5 2.5×102.57 2.5×102.5 3000003 3000003 3000003

5 7 7 5 5 2.0×102.0 2.0×102.07 2.0×102.0 7 7 * 7 * * 7 * 7 * 7 * * 2000002 1.5×101.5 2000002 1.5×101.5 * 2000002 1.5×101.5 * * 1.0×107 7 1.0×107 RLUs x10 1.0×10 RLUs x10 RLUs RLUs x10 1.0 * 1.0 1.0 RLUs x10 RLUs x10 RLUs x10 * 1000001 1000001 100000* 1 5.0×100.56 5.0×100.56 5.0×100.56 * 0 0.00 0 0.00 0 0.00 01050g/ml 01050g/ml g/ml 010255001050g/mlg/ml 0102550g/ml 0102550 Figure 4. HNP1, HD5 and RC101 inhibit infection by pseudotyped SARS-CoV-2 variants. Pseudotyped luciferase reporter Figure 4. HNP1, HD5B and B.1.1.7 RC101 variant inhibit (UK) infection by pseudotyped SARS‐CoV‐2 variants. Pseudo‐ virus expressing SARS-CoV-2 S protein from the P.1 variant (A) or the B.1.1.7 variant (B) was incubated with or without typed luciferase reporter virus expressing SARS‐CoV‐2 S protein from the P.1 variant (A) or the HNP1, HD5 and RC101 at 37 ◦C for400000 14 h. Viruses wereHNP1 added400000 to HeLa-hACE24 HD5 cells and incubated4000004 at 37 ◦C RC101 for 2 h. Infected B.1.1.7 variant (B) was incubated with or without HNP1, HD5 and RC101 at 37 °C for 1 h. Viruses cells were cultured for two days before3000003 measuring luciferase300000 activity.3 The significance of the300000 differences3 between defensin- 5 5 were added to HeLa‐hACE25 cells and incubated at 37 °C for 2 h. Infected cells were cultured for two treated virions and mocked-treated controls was* calculated by Student’s two-tailed, unpaired t-test; * p < 0.05. Data are * * * 2000002 * 2000002 2000002 means ± SD and are representative of three independent experiments. * * RLUs x10 RLUs x10 RLUs RLUs x10 * 1000001 1000001 1000001 3.2. Defensins Inhibit Viral Entry 0 0 0 We determined0102550 whetherg/ml pre-treatment0102550 of cellsg/ml with defensins0102550 provided protectiong/ml against the virus. Cells were treated with defensins for 1 h, washed and then infected withFigure pseudotyped 4. HNP1, HD5 SARS-CoV-2 and RC101 inhibit virus. infection Pretreatment by pseudotyped of cells with SARS high‐CoV concentrations‐2 variants. Pseudo of ‐ HNP1,typed luciferase HD5 and reporter RC-101 partiallyvirus expressing blocked SARS viral‐CoV infection‐2 S protein (Figure from5A). the To determineP.1 variant whether(A) or the defensinsB.1.1.7 variant exhibited (B) was an incubated effect after with viral or without entry, cells HNP1, were HD5 infected and RC101 with at pseudotyped 37 °C for 1 h. SARS-Viruses CoV-2were added viruses to forHeLa 1.5‐hACE2 h and cells then and treated incubated with HNP1,at 37 °C HD5for 2 h. or Infected RC101 cells for three were days.cultured None for two of

Viruses 2021, 13, x FOR PEER REVIEW 8 of 15

days before measuring luciferase activity. The significance of the differences between defensin‐ treated virions and mocked‐treated controls was calculated by Student’s two‐tailed, unpaired t‐test; * p < 0.05. Data are means ± SD and are representative of three independent experiments.

3.2. Defensins Inhibit Viral Entry We determined whether pre‐treatment of cells with defensins provided protection against the virus. Cells were treated with defensins for 1 h, washed and then infected with pseudotyped SARS‐CoV‐2 virus. Pretreatment of cells with high concentrations of HNP1, HD5 and RC‐101 partially blocked viral infection (Figure 5A). To determine whether de‐ Viruses 2021, 13, 1246 8 of 15 fensins exhibited an effect after viral entry, cells were infected with pseudotyped SARS‐ CoV‐2 viruses for 1.5 h and then treated with HNP1, HD5 or RC101 for three days. None of the tested defensins affected infection after viral entry (Figure 5B). These results sug‐ gestedthe tested that defensins affected block SARS infection‐CoV after‐2 infection viral entry at (Figure the step5B). of These viral results entry. suggested that defensins block SARS-CoV-2 infection at the step of viral entry.

A Pretreatment of cells before viral infection

800008 HNP1 600006 HD5 400004 RC101

600006 300003 4 4 * 400004 4 400004 * * 200002 * 2 * * RLUs x10 RLUs x10 RLUs

20000 RLUs x10 200002 100001

0 0 0 0 1 12.5 25 50 g/ml 0 1 12.5 25 50 g/ml 0 1 12.5 25 50 g/ml

B Treatment after viral entry

HNP1 HD5 RC101 2000020 800008 600006

1500015 600006 3 4 4 400004

1000010 400004 2 RLUs x10 20000 RLUs x10 RLUs RLUs x10 50005 200002

0 0 0 0 1 12.5 25 g/ml 0 1 12.5 25 50 g/ml 0 1 12 25 50 g/ml

FigureFigure 5. Defensins 5. Defensins are ineffective are ineffective after after viral viral entry entry.. (A ()A To) To determine determine the the effect ofof defensinsdefensins on on target target cell cell resistance resistance to infection,to infection, HEK293T HEK293T-hACE2‐hACE2 cells were cells incubated were incubated with withdefensins defensins in the in presence the presence of FBS of FBS for 1 for h, 1and h, and then then exposed exposed to pseudo to ‐ typedpseudotyped SARS‐CoV‐ SARS-CoV-22 luciferase luciferasereporter virus reporter without virus without defensins defensins for 2 h. for Cells 2 h. Cellswere were washed washed and and then then cultured cultured in in fresh fresh media for threemedia days for before three days measuring before measuring luciferase luciferase activity. activity. (B) To determine (B) To determine the post the‐entry post-entry effect effect of defensins, of defensins, HEK293T HEK293T-‐hACE2 cells werehACE2 infected cells were with infected pseudotyped with pseudotyped SARS‐CoV SARS-CoV-2‐2 viruses for viruses 2 h followed for 2 h by followed treatment by treatment of infected of infectedcells with cells the with indicated concentrationthe indicated of defensins. concentration The of differences defensins. The between differences defensin between‐treated defensin-treated samples and samples untreated and untreatedcontrols were controls calculated were by Student’scalculated two‐tailed by Student’s t‐test; * two-tailed p < 0.05. Datat-test; are * p means< 0.05. ± Data SD of are triplicate means ± samplesSD of triplicate and are samples representative and are representative of three independent of experiments.three independent experiments. The step of viral entry includes viral attachment and the binding of spike proteins throughThe step the RBDof viral to hACE2, entry followedincludes by viral fusion attachment between the and viral the and binding cellular of membranes. spike proteins throughTo assess the the RBD effect to hACE2, of HNP1 followed on viral attachment, by fusion between pseudotyped the viral SARS-CoV-2 and cellular viruses membranes. with Toor assess without the HNP1effect of pretreatment HNP1 on viral were attachment, incubated with pseudotyped HeLa-hACE2 SARS cells‐CoV at 4 ◦‐C2 viruses for 2 h. with or Afterwithout washing HNP1 to removepretreatment unbound were viruses, incubated cells were with lysed, HeLa and‐ thehACE2 level ofcells cell-associated at 4 °C for 2 h. AfterHIVp24 washing was determined.to remove unbound HNP1 did viruses, not have cells a significant were lysed, impact and on the viral level attachment of cell‐associ‐ atedexcept HIVp24 that was the high determined. concentration HNP1 (50 didµg/mL) not have of HNP1 a significant suppressed impact viral on attachment viral attachment by except35% (Figurethat the6A). high We concentration did not find that (50 HNP1 μg/mL) interfered of HNP1 with thesuppressed interaction viral between attachment spike by RBD and hACE2 proteins (Figure6B). In contrast, HNP1 at high concentrations (16.7 and 50 µg/mL) slightly promoted the interaction between hACE2 and spike RBD proteins. We then assessed the effect of HNP1 on viral fusion. Pseudotyped viruses expressing SARS- CoV-2 spike D614G proteins (in the UK B.1.1.7 variant) were packaged with BlaM-Vpr. Virus binding to cells was augmented by low-speed centrifugation at 16 ◦C. After washing off unbound viruses, cells were incubated in medium with or without defensins at 37 ◦C to allow viral fusion to occur. In the absence of defensins, β-lactamase, released into the cytoplasm from the viruses containing BlaM-Vpr proteins, cleaved the CCF4-AM substrate Viruses 2021, 13, x FOR PEER REVIEW 9 of 15

35% (Figure 6A). We did not find that HNP1 interfered with the interaction between spike RBD and hACE2 proteins (Figure 6B). In contrast, HNP1 at high concentrations (16.7 and 50 μg/mL) slightly promoted the interaction between hACE2 and spike RBD proteins. We then assessed the effect of HNP1 on viral fusion. Pseudotyped viruses expressing SARS‐ CoV‐2 spike D614G proteins (in the UK B.1.1.7 variant) were packaged with BlaM‐Vpr. Virus binding to cells was augmented by low‐speed centrifugation at 16 °C. After washing off unbound viruses, cells were incubated in medium with or without defensins at 37 °C Viruses 2021, 13, 1246to allow viral fusion to occur. In the absence of defensins, β‐lactamase, released into the 9 of 15 cytoplasm from the viruses containing BlaM‐Vpr proteins, cleaved the CCF4‐AM sub‐ strate (Figure 6C, left panel), resulting in an increase in fluorescent signals at 460 nm. We found that HNP1(Figure blocked6C, left viral panel), fusion resulting in ina dose an increase‐dependent in fluorescent manner signals (Figure at 460 6C, nm. right We found panel). that HNP1 blocked viral fusion in a dose-dependent manner (Figure6C, right panel).

A Viral attachment B RBD-hACE binding

4008 2.0 *

3006 1.5 * * 2004 1.0 O.D. 405 nm HIV p24 pg/ml pg/ml p24 HIV 1002 0.5 HIV p24 ng/ml

00 0.0 0 5 25 50 g/ml 0 0.6 1.9 5.6 16.7 50 g/ml C Fusion

Pseudotyped SARS-CoV-2 virus containing BlaM-Vpr

120

BlaM-Vpr 80 520nm 520nm low * high 40 cleave Fusion 460 nm 460 nm Low High of control) (% BlaM substrate 0 012.52550g/ml 410nm Fusion signal F460/F520

Figure 6. The effect of HNP1 on viral attachment, spike RBD-hACE2 interaction and viral fusion. (A) Pseudotyped SARS- CoV-2 virusesFigure were 6. treatedThe effect with of HNP1 HNP1 at differenton viral concentrations attachment, spike for 1 h RBD at 37‐hACE2◦C. The virus-defensininteraction and mixture viral wasfusion. added to HeLa-hACE2(A) Pseudotyped cells, and cells SARS were‐CoV incubated‐2 viruses at 4 were◦C for treated 2 h. After with washing HNP1 off at unbounddifferent viruses,concentrations cells were for lysed, 1 h at and cell-associated37 °C. HIVp24 The virus was‐ determineddefensin mixture as described was added in the Methods. to HeLa (‐BhACE2) Immobilized cells, and spike cells RBD were proteins incubated on the plateat 4 °C were incubatedfor with 2 h. biotinylated After washing hACE off proteins unbound that viruses, were pre-treated cells were with lysed, defensins and cell at different‐associated concentrations HIVp24 was for 1deter h. After‐ incubationmined at 37 ◦ asC fordescribed 1 h, the platein the was Methods. washed ( andB) Immobilized the bound hACE2 spike proteins RBD proteins were detected on the by plate incubation were incubated with alkylate phosphatasewith (AP)-conjugated biotinylated hACE streptavidin proteins followed that were by the pre AP‐treated colorimetric with assay defensins as described at different in the concentrations Methods. For panels for A and B, the1 differences h. After incubation between defensin-treated at 37 °C for 1 samplesh, the plate and untreatedwas washed controls and were the bound calculated hACE2 by Student’s proteins two-tailed were t-test; * pdetected< 0.05. Data by incubation are means ±withSD alkylate of triplicate phosphatase samples and (AP) are‐conjugated representative streptavidin of three independent followed by experiments. the AP (C) A schematiccolorimetric of Förster assay resonance as described energy in transfer the Methods. (FRET)-based For panels BlaM assayA and is B, shown the differences on the left panel. between Pseudotyped defen‐ SARS-CoV-2sin‐ virusestreated containing samples BlaM-Vprand untreated proteins controls were incubated were calculated with Huh7.5-ACE2-TMPRSS2 by Student’s two‐tailed cells at t‐ 16test;◦C * for p < 30 0.05. min at 1550× g. AfterData washingare means off ± unbound SD of triplicate viruses, samples cells were and treated are representative with HNP1 at different of three concentrations independent andexperiments. incubated at (C) A schematic of Förster resonance energy transfer (FRET)‐based BlaM assay is shown on the left 37 ◦C for 2 h for viral fusion. The BlaM substrate, CCF4-AM, was added to cells, which were incubated at 11oC overnight. panel. Pseudotyped SARS‐CoV‐2 viruses containing BlaM‐Vpr proteins were incubated with The cytoplasmic BlaM activity from viral fusion was determined by measuring the ratio of blue (460 nm) to green (520 nm) Huh7.5‐ACE2‐TMPRSS2 cells at 16 °C for 30 min at 1550× g. After washing off unbound viruses, fluorescence with excitation at 410 nm. The differences between defensin-treated samples and untreated controls were cells were treated with HNP1 at different concentrations and incubated at 37 °C for 2 h for viral calculated by Student’s two-tailed t-test; * p < 0.05. Data are means ± SD of two independent experiments. fusion. The BlaM substrate, CCF4‐AM, was added to cells, which were incubated at 11oC overnight. The cytoplasmic 3.3.BlaM Native activity Structure from viral (Disulfide fusion Bonding) was determined of Defensins by measuring Is Required the to Inhibit ratio of blue (460 nm) to green (520SARS-CoV-2 nm) fluorescence Infection with excitation at 410 nm. The differences between defensin‐ The structure of defensins is critical for their effects on viruses [16,39]. To determine whether this was true of SARS-CoV-2 as well, we examined the anti-SARS-CoV-2 activity of [Abu]HNP1 and [Abu]HD5, which are identically charged, but unstructured analogs of HNP1 and HD5 [22]. We treated pseudotyped SARS-CoV-2 virus with these linear defensins before adding the virus to cells and then determined infection at day three Viruses 2021, 13, x FOR PEER REVIEW 10 of 15

treated samples and untreated controls were calculated by Student’s two‐tailed t‐test; * p < 0.05. Data are means ± SD of two independent experiments.

3.3. Native Structure ( Bonding) of Defensins Is Required to Inhibit SARS‐CoV‐2 In‐ fection The structure of defensins is critical for their effects on viruses [16,39]. To determine whether this was true of SARS‐CoV‐2 as well, we examined the anti‐SARS‐CoV‐2 activity of [Abu]HNP1 and [Abu]HD5, which are identically charged, but unstructured analogs Viruses 2021, 13, 1246 of HNP1 and HD5 [22]. We treated pseudotyped SARS‐CoV‐2 virus with these10 linear of 15 de‐ fensins before adding the virus to cells and then determined infection at day three post‐ infection. In contrast to the antiviral activity of HNP1 or HD5 (Figure 1), the linear analogs [Abu]HNP1post-infection. and In[Abu]HD5 contrast to did the antiviralnot have activity an inhibitory of HNP1 effect or HD5 on (Figure SARS1‐),CoV the‐ linear2 infection (Figureanalogs 7A). [Abu]HNP1 These results and indicate [Abu]HD5 that did the not antiviral have an effect inhibitory of defensins effect on requires SARS-CoV-2 native di‐ sulfideinfection bonding (Figure and7A). Theseproperly results folded indicate proteins. that the HD5 antiviral is effectsynthesized of defensins as a requires propeptide (ProHD5)native disulfide in Paneth bonding cells andin the properly small folded intestine proteins. and in HD5 epithelial is synthesized cells of as athe propeptide genital tract, and(ProHD5) is processed in Paneth to the cells mature in the smallpeptide intestine by trypsin and in or epithelial neutrophil cells ofproteases the genital [34,40]. tract, The unprocessedand is processed pro‐HD5 to the exhibited mature peptide no anti by‐SARS trypsin‐CoV or neutrophil‐2 activity proteases(Figure 7B), [34,40 which]. The is in agreementunprocessed with pro-HD5 prior studies exhibited reporting no anti-SARS-CoV-2 reduced or activity absent (Figure antimicrobial7B), which activity is in of agreement with prior studies reporting reduced or absent antimicrobial activity of proHD5 proHD5 compared to processed HD5 [34,40]. compared to processed HD5 [34,40].

A [Abu] HNP1 [Abu] HD5 B ProHD5 500005 20000020 25000025

400004 20000020 15000015 4 4 3 300003 15000015 10000010 200002 10000010 RLUs x10 RLUs x10 RLUs x10 500005 100001 500005

0 0 0 0 1 12.5 25 50 g/ml 025g/ml 02550100g/ml

Figure 7. The structure of defensins required for anti-SARS-CoV-2 activity. To determine the effect of linear defensins Figure(A 7.) orThe proHD5 structure (B) onof defensins viral infection, required pseudotyped for anti‐SARS SARS-CoV-2‐CoV‐2 virus activity. was To incubated determine with the indicated effect of concentrations linear defensins of (A) or proHD5[Abu]HNP1 (B) on or [Abu]HD5viral infection, for 1 h pseudotyped before infection SARS of HEK293T-hACE2‐CoV‐2 virus was cells. incubated Infected cells with were indicated cultured forconcentrations three days of [Abu]HNP1before measuring or [Abu]HD5 luciferase for 1 activity. h before There infection was no of difference HEK293T between‐hACE2 defensin-treated cells. Infected samplescells were and cultured untreated for controls three days beforeas measuring calculated luciferase by Student’s activity. two-tailed Theret-test. was Datano difference are means between± SD of defensin triplicate‐treated samples samples and are representativeand untreated of controls two as calculatedindependent by Student’s experiments. two‐tailed t‐test. Data are means ± SD of triplicate samples and are representative of two inde‐ pendent experiments. 3.4. The effect of Virus Titers and Cell Types on Defensin-Mediated Viral Infection 3.4. TheWe effect have of previouslyVirus Titers shown and Cell that Types the direct on Defensin effect of HNP1‐Mediated on HIV Viral virions Infection was abolished by an increase in the number of virus particles [30]. To determine whether increasing the virusWe titer have affected previously the antiviral shown activity that the of HNP1, direct different effect of titers HNP1 of serum-free on HIV virions pseudotyped was abol‐ ishedSARS-CoV-2 by an increase were incubated in the number with 1 of or virus 25 µg/mL particles HNP1 [30]. at 37 To◦C determine for 1 h and whether then added increas‐ ingto the cells. virus Viral titer infection affected was the determined antiviral at activity day three of post-infection. HNP1, different At 25 titersµg/mL, of HNP1serum‐free pseudotypedinhibited viral SARS infection‐CoV regardless‐2 were incubated of the virus with titer. 1 At or 1 25µg/mL, μg/mL however, HNP1 HNP1 at 37 displayed°C for 1 h and thenan added inhibitory to effectcells. onViral low infection concentrations was determined of the virus, butat day this effectthree was post lost‐infection. when the At 25 μg/mL,viral titerHNP1 was inhibited high (Figure viral8 A).infection This result regardless suggest of that the HNP1virus titer. interacts At 1 directly μg/mL, with however, the virus, and is depleted at high viral concentrations (Figure8A). Finally, we examined HNP1 displayed an inhibitory effect on low concentrations of the virus, but this effect was whether the SARS-CoV-2 inhibitory effect of defensins was cell-type-dependent. Intestinal lostepithelial when the cells viral (Caco-2) titer express was high moderate (Figure levels 8A). of endogenousThis result hACE2,suggest and that lung HNP1 epithelial interacts directlycells (A549) with the have virus, near undetectable and is depleted levels at of high hACE2 viral but concentrations a high abundance (Figure of SARS-CoV-2 8A). Finally, wealternative examined receptors, whether CD147the SARS and‐CoV tyrosine-protein‐2 inhibitory kinase effect receptor of defensins UFO (AXL) was [cell28].‐type We ‐de‐ pendent.found thatIntestinal HNP1, HD5epithelial and RC101 cells exhibited (Caco‐ anti-SARS-CoV-22) express moderate activity levels in both of Caco-2 endogenous and hACE2,A549 cellsand lung (Figure epithelial8B), indicating cells (A549) that the have inhibitory near undetectable effect of defensins levels on of viral hACE2 infection but a high abundancewas not receptor-specific of SARS‐CoV‐ or2 alternative cell-type-dependent. receptors, CD147 and tyrosine‐protein kinase re‐ ceptor UFO (AXL) [28]. We found that HNP1, HD5 and RC101 exhibited anti‐SARS‐CoV‐

Viruses 2021, 13, x FOR PEER REVIEW 11 of 15

Viruses 2021, 13, 1246 2 activity in both Caco‐2 and A549 cells (Figure 8B), indicating that the inhibitory effect11 of 15of defensins on viral infection was not receptor‐specific or cell‐type‐dependent.

A B HNP1 1 g/ml HNP1 25 g/ml Intestinal Lung 200 100 40000 30003 4 epithelial cells epithelial cells 80 150 300003 3 4 20002 60 100 * * 200002 40

1000RLUs x10 RLUs x10 %of control

% of control control of % 1 50 100001 * 20 * * * * * 0 0 0 0 1:0 1:1 1:2 1:3 1:0 1:1 1:2 1:3 l controlHNP1 HD5 RC101 Controlo HNP11 HD5 RC101 Control HNP1 HD5 RC101 tr P 5 1 n N HD 10 Virus dilution Virus dilution o H RC c FigureFigure 8. 8. TheThe effecteffect ofof viralviral titerstiters andand cellcell typestypes onon defensin-mediateddefensin‐mediated SARS-CoV-2SARS‐CoV‐2 inhibition.inhibition. ((AA)) ToTo determine determine the the effect effect ofof virus virus titers titers on on the the antiviral antiviral activity activity ofof HNP1,HNP1, indicatedindicated dilutionsdilutions ofof serum-freeserum‐free pseudotypedpseudotyped SARS-CoV-2SARS‐CoV‐2 viruses viruses were were ◦ incubatedincubated withwith 11µ μg/mLg/mL ((leftleft)) oror 2525 μµg/mLg/mL (right) HNP1 at 37 °CC forfor 11 hh beforebefore infectioninfection ofof HEK293T-hACE2 HEK293T‐hACE2 cells. cells. TheThe percentagepercentage of of control control was was calculated calculated using using thethe formula:formula: (RLU(RLU ofof defensin-treateddefensin‐treated samples/RLUssamples / RLUs of of average average of of untreated untreated samplessamples atat thethe same virus titer) titer) x× 100.100. (B ()B To) To determine determine the the effect effect of ofdefensins defensins on onSARS SARS-CoV-2‐CoV‐2 infection infection of intestinal of intestinal and andlung lung epithelial epithelial cells cellsexpressing expressing endogenous endogenous hACE2 hACE2 receptors, receptors, pseudotyped pseudotyped SARS‐CoV SARS-CoV-2‐2 viruses viruses were incubated were incubated with 25 withμg/mL 25 HNP1,µg/mL HD5 HNP1, or RC101 HD5 or at RC101 37 °C for at 371 h,◦C then for added 1 h, then to intestinal added to intestinalCaCo‐2 cells CaCo-2 (left) or cells lung (left epithelial) or lung A549 epithelial cells ( A549right) at 37 °C for 1.5 h. After washing off unbound viruses, infected cells were cultured for three days before measuring lucif‐ cells (right) at 37 ◦C for 1.5 h. After washing off unbound viruses, infected cells were cultured for three days before erase activities. The differences between defensin‐treated samples and untreated controls were calculated using Student’s measuring luciferase activities. The differences between defensin-treated samples and untreated controls were calculated two‐tailed t‐test; * p < 0.05. Data are means ± SD of triplicate samples and are representative of three independent experi‐ using Student’s two-tailed t-test; * p < 0.05. Data are means ± SD of triplicate samples and are representative of three ments. independent experiments.

4.4. DiscussionDiscussion TheThe abilityability ofof thethe innateinnate immuneimmune responseresponse toto reducereduce viralviral replicationreplication isis knownknown toto impactimpact thethe severityseverity ofof coronavirus-associatedcoronavirus‐associated diseasedisease outcomes.outcomes. DefensinsDefensins areare highlyhighly abundantabundant antimicrobial peptides produced by by neutrophils neutrophils and and epithelial epithelial cells, cells, and and play play a acritical critical role role in in innate innate immunity. immunity. Here, Here, we we showed that humanhuman α‐α-defensinsdefensins includingincluding HNPs1–3HNPs1–3 andand HD5HD5 asas well as θ‐θ-defensindefensin analog RC-101RC‐101 suppressed SARS-CoV-2SARS‐CoV‐2 infectioninfection atat thethe stepstep ofof viralviral entry.entry. HNP1,HNP1, HD5HD5 andand RC101RC101 alsoalso blockedblocked infectioninfection by the BrazilBrazil P.1 andand thethe UKUK B.1.1.7B.1.1.7 variants.variants. Linear unstructured defensins did not inhibit viral infection. Furthermore,Furthermore, thethe antiviralantiviral activityactivity ofof defensinsdefensins waswas alsoalso foundfound forfor infectionsinfections ofof epithelialepithelial cellscells whichwhich diddid notnot overexpressoverexpress hACE2.hACE2. OurOur resultsresults showedshowed thatthat thethe anti-SARS-activityanti‐SARS‐activity ofof defensinsdefensins waswas specificspecific forfor defensindefensin subtypes.subtypes. HBDsHBDs are are generally generally more more cationic cationic and and less less hydrophobic hydrophobic than than human humanα-defensins. α‐defen‐ Differentialsins. Differential anti-SARS-CoV-2 anti‐SARS‐CoV activities‐2 activities among among HBDs HBDs and lectin-like and lectin HNPs1–3,‐like HNPs1–3, HD5 HD5 and RC101and RC101 suggest suggest that hydrophobic that hydrophobic binding binding properties properties of these of defensins these defensins may play may a critical play a rolecritical in theirrole in viral their inhibition. viral inhibition. ProHD5 ProHD5 exhibits exhibits reduced reduced lectin-like lectin‐like properties, properties, which which is consistentis consistent with with the the fact fact that that the the pro-region pro‐region functionally functionally inhibits inhibits defensin defensin activity,activity, andand presumablypresumably contributescontributes toto thethe lossloss ofof antiviralantiviral activityactivity [[34].34]. WeWe alsoalso foundfound differentialdifferential anti-SARS-CoV-2anti‐SARS‐CoV‐2 activitiesactivities betweenbetween HD5HD5 andand HD6HD6 (Figure(Figure1 1),), which which is is not not surprising surprising as as thesethese twotwo intestinalintestinal defensinsdefensins exhibitexhibit distinctdistinct structuresstructures andand properties,properties, particularly particularly in in the the casecase ofof glycan-mediatedglycan‐mediated viralviral attachmentattachment [[7,41].7,41]. DetailedDetailed mechanismsmechanisms ofof defensin-mediateddefensin‐mediated inhibitioninhibition ofof SARS-CoV-2SARS‐CoV‐2 fusionfusion remainremain toto bebe determined.determined. Analyses of of specific specific steps steps during during viral viral entry entry revealed revealed that that HNP1 HNP1 had had a aweak weak inhibitory inhibitory effect effect on on viral viral attachment attachment and and no effect no effect on the on binding the binding of the of spike the spike RBD RBDdomain domain to hACE2, to hACE2, but significantly but significantly inhibited inhibited SARS SARS-CoV-2‐CoV‐2 spike spike protein protein-mediated‐mediated viral viralfusion. fusion. Defensins Defensins have also have been also shown been shown to block to viral block fusion viral of fusion HIV of[13,42], HIV [but13,42 specific], but specificmechanisms mechanisms of action of were action not were reported. not reported. Defensins Defensins may alter may protein alter protein domains domains of spike of spikeproteins proteins important important for viral for fusion viral fusion [43]. Additionally, [43]. Additionally, these these peptides peptides may maysuppress suppress viral viral entry by aggregating virions [44] or by affecting the bilayer structure of the viral membrane. Several host factors including TMPRSS2 and TMPRSS4, neutrophilin-1 and heparan sulfate interact with spike proteins and promote SARS-CoV-2 infection [45–50]. Thus, Viruses 2021, 13, 1246 12 of 15

defensins may inhibit viral entry by acting on these host factors. For example, TMPRSS2 has been shown to prime SARS-CoV-2 proteins for hACE2-dependent viral entry [45]. Since RC101 is known to inhibit Japanese encephalitis virus protease [51], investigations into the effects of defensins on serine protease are warranted. Additionally, alternative receptors including CD147 and AXL for SARS-CoV-2 viral entry have been reported, particularly in cells with a low abundance of hACE2 [28,52,53]. We have shown that pseudotyped viruses expressing SARS-CoV-2 spike proteins enter A549 cells through CD147 in a spike RBD-independent manner [28]. Similarly, AXL mediates SARS-CoV-2 infection of lung epithelial cells through interactions with the N-terminal domain of SARS- CoV-2 spike proteins [53]. We found that HNP1 did not impact the binding of RBD to hACE2 but suppressed pseudotyped SARS-CoV-2 infection of A549 cells, suggesting that defensins may interfere with the binding of spike proteins to alternative receptors such as CD147. Defensins are known for exhibiting both pro-inflammatory and anti-inflammatory activities, and they can suppress viral infection by modulating immune cell activities [14,19]. Therefore, although HBDs did not block viral entry, these peptides may inhibit viruses by promoting host restriction factors in specific cell types. Studies on the role of defensins in the regulation of viral infection through immune modulation using innate immune cells will offer insights into immune-modulatory functions of defensins relevant to SARS-CoV-2 infection and disease outcomes. Defensins represent one category of antimicrobial peptides [54]. Antimicrobial pep- tides are evolutionarily conserved molecules for defense, which are produced by , insects, and [54,55]. More than 3000 antimicrobial peptides have been reported [56]. Considering their versatile properties including anti-microbial activities and immune functions, the development of natural or synthetic antimicrobial peptides against SARS-CoV-2 and other viruses may help combat emerging in the future. Our findings have clinical relevance. Individuals differ in their defensin repertoire, which may influence disease outcomes in response to SARS-CoV-2 infection [57–60]. Older adults and people with hypertension have dysfunctional neutrophils [61–64], and they have higher mortality in response to SARS-CoV-2 infection [65–68]. It is possible that in these high-risk groups, defensin levels are insufficient to block virus replication during acute infection and are insufficient to control inflammation mediated by in the aftermath of infection, which may provide a partial explanation for severe disease outcomes in these groups. Furthermore, HNPs form stable complexes with lipoprotein (a) and low-density lipoproteins, which are elevated in people with hypertension [69]. Thus, studies on the effects of lipoproteins on HNP-mediated SARS-CoV-2 inhibition will likely offer insights into COVID-19 disease pathogenesis. In summary, our findings provide insights into the function of human α-defensins in innate immunity against SARS-CoV-2 infection. Our findings show that α-defensins and their analog RC101 are inhibitors of SARS-CoV-2 infection. Understanding the mechanisms by which defensins and their analogs block SARS-CoV-2 infection offers new therapeu- tic avenues.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/v13071246/s1, Figure S1: Defensins with anti-SARS-CoV2 activity are not cytotoxic. Author Contributions: Conceptualization, W.L. and T.L.C.; investigation, C.X., A.W., M.M., W.H., S.R., S.S., A.P., G.B.M., W.L. and T.L.C.; data curation, C.X., A.W., M.M., S.R, W.H. and T.L.C.; writing— original draft preparation, C.X., W.H., M.M. and T.L.C.; writing—review and editing, C.X., A.W., M.M., W.H., S.R., E.P., S.S., A.P., G.B.M., W.L. and T.L.C.; supervision, T.L.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by NIH grants R01AI36948 (T.L.C.) and R01AI053668 (G.B.M.), and by the National Natural Science Foundation of China’s grant 82030062 (W.L.). Institutional Review Board Statement: Not applicable. Viruses 2021, 13, 1246 13 of 15

Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank Eric B. Milner for editing the manuscript. Conflicts of Interest: The authors declare that there is no conflict of interest.

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