*2.2. Antiviral Activity of Defensins*

The antiviral activity of defensins was first reported in 1986 [10]. Since then, defensins have demonstrated protection against human immunodeficiency virus (HIV), influenza A virus (IAV), human adenovirus (HAdV), severe acute respiratory syndrome coronavirus (SARSC), papillomavirus (HPV), respiratory syncytial virus (RSV), and herpes simplex virus (HSV) [5,10,15–18]. Recent studies have focused on elucidating the multiple mechanisms associated with defensins' antiviral activity (Table 1). Defensins can block viral infection through direct action on virus particles or interfere indirectly at various stages of the viral life cycle [10,18]. Available data suggest antiviral activity occurs predominantly at viral entry steps; however, antiviral effects at other stages of infection have also been reported, particularly affecting viral trafficking within infected cells [19]. Defensins can also modify the innate immune response to viral infections, including: modulation of T-cells, macrophage and dendritic cells recruitment to sites of infection, wound healing and angiogenesis, differentiation and maturation of dendritic cells, induction of the production of pro-inflammatory cytokines by macrophages, mast cells, and keratinocytes, and regulation of cell death pathways [9]. For example, HBD-3 can suppress activation of the caspase cascade to prevent apoptosis in infected cells [20]. Similarly, the concentration of HNPs released into the microenvironment upon activation of neutrophils during inflammation exerts a differential effect on cytokine production in activated monocytes [19]. HNP concentrations of 1 to 10 nM can upregulate the expression of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), whereas concentrations of 10 to 100 μM are cytotoxic to monocytes.

#### *2.3. Adenovirus*

Human adenovirus (HAdV) is a non-enveloped double-stranded DNA virus that is capable of infecting the respiratory, gastrointestinal, ocular, and excretory systems in humans. There are approximately 80 recognized HAdV serotypes, subdivided into species A–G [21,22]. Currently there are only a limited number of HAdV therapeutic strategies and vaccines available to treat HAdV infections. Alpha defensins have demonstrated an ability to hinder HAdV infections in vitro [21,23]. HD5 reduces HAdV replication by 95% when cells are exposed to the peptide (IC50 = 3–4 μM) prior to infection, and by 50% when peptide is added 30–60 min post inoculation, suggesting that inhibition occurs at an early stage during viral infection [21]. Additional studies have shown that direct binding of HD5 (10 μM) to HAdV particles prior to infection prevents the release of internalized viral particles from endosomes [24]. Subsequently, viral particles appear to colocalize with lysosomes indicating altered viral trafficking following infection as a consequence of HD5 binding [24]. These findings suggest defensins' antiviral activity against HAdV results in blockage of HAdV uncoating and genome exposure [24]. In addition, HD5 antiviral activity is species specific; pre-treatment with 15μM HD5 decreased HAdV infectivity of subspecies A–C and E, while infectivity of HAdV subspecies D and F demonstrated no change [23]. The cause of species specificity of HD5 activity is yet to be determined.

#### *2.4. Influenza A Virus*

During the early infiltrate in influenza A virus (IAV), neutrophils predominate in infected airways, highlighting their importance in initiating immune responses against IAV [13]. Defensins are; thus, likely to interact with IAV. Neutrophil extracellular traps (NETs) displaying HNPs are formed in vivo and in vitro in response to IAV infection [13]. Cells incubated with defensins pre- or post-infection demonstrated minimal inhibitory activity against IAV, whereas incubation of HNPs with virions prior to infection is necessary for the antiviral activity of these AMPs against IAV (e.g., HNP1 IC50 < 2 μg/mL [25]) [13,26]. In addition, the binding activity of defensins against IAV is increased by formation of multi-molecular assemblies of defensins, which may be responsible for pore formation in the IAV envelope, thereby destabilizing virions prior to receptor binding and cellular entry [13].

Expression of β-defensins HBD1, HBD2, and HBD3 has been reported in various epithelial cell tissues, with each β-defensin demonstrating a unique expression induction profile in response to IAV infection [13]. However, HBD1 and HBD2 have also been detected in monocytes, macrophages and monocyte-derived dendritic cells (DCs), and possess strong neutralizing activity against multiple IAV strains [10,13]. HBDs exhibit low potency as direct inhibitors of IAV virions, but are speculated to play important immunomodulatory roles by limiting inflammation during IAV infection [13]. While the exact sequence of immunomodulatory events is yet to be determined, it has been reported that deletion of the HBD1 analog in mice resulted in a more serious inflammatory reaction to IAV [13,27]. In addition, HBD3 has demonstrated strong anti-inflammatory effects in cells stimulated with 50 ng/mL lipopolysaccharide (LPS), confirmed by the inhibition of expression of inflammatory mediators such as (TNF-α) [28]. Conversely, α-defensins inhibit IAV replication in infected cells. Pre-incubation of virions with HNP-1 (25 μg/mL) is capable of reducing the replication of IAV strain H1N1 by 10- to 1000-fold in multiple cell lines when compared to replication in untreated cells [29]. Similarly, HNP-1 and 2 can reduce infectious virus of the Phil82 strain of IAV by 85% to 90% in various cell lines [26].

#### *2.5. Human Immunodeficiency Virus*

Defensins demonstrate antiviral activity against human immunodeficiency virus (HIV), mediated by direct virus–peptide interaction and/or inhibition of viral genome replication. Inhibition mediated by the direct binding of defensins with HIV virions is attributed to interactions between positively-charged HNPs and negatively-charged moieties of the HIV envelope glycoprotein gp120. HNP1, HNP2, and

HNP3 function as lectins by directly blocking the interaction of gp120 and the HIV receptor CD4. However, the exact mechanism of this interaction is not well characterized [10,13]. HNP-1 can also interfere with critical steps in the HIV replication cycle [30]. HNP-1 inhibits protein kinase C signaling, which is important for the transcription and nuclear import of the HIV genome [31]. HD5 exhibits a robust dose-dependent (IC50 = 400 nM) suppression of HIV-1 replication in absence of serum when pre-incubated with virions [32]. HD5 also blocks HIV-1 infection at a step prior to viral entry [32]. HD5 competitively binds to the CD4 receptor in a dose-dependent manner against HIV, thereby blocking HIV entry into target cells [32]. Interestingly, in contrast to HNP1, HNP2, HNP3, and HD5, HNP4 does not interact with CD4 or HIV gp120 [13]. HNP4 inhibits HIV replication with greater effectiveness than HNP1, HNP2, and HNP3, but it is unclear whether efficacy of HNP4 is mediated only through a direct effect on virions or also on host processes that ultimately affect viral replication.

β-defensins can also exert antiviral activities against HIV. Expression of HBD2 and HBD3 can be induced by microbial products such as endotoxins, viruses, bacteria, and pro-inflammatory cytokines such as TNF and IL-1β [13]. Expression of HBD2 and HBD3, but not HBD1, mRNA can be induced by HIV in human oral epithelial cells. HBD2 inhibits the formation of early HIV transcript products but does not affect cell–cell fusion [10].

#### *2.6. Herpes Simplex Virus*

Alpha and β-defensins can exhibit anti-HSV properties [33]. HNP-1-4 and HD6 inhibit HSV binding to its target receptor by directly interacting with either the HSV glycoprotein or by binding to heparan sulfate (HS), thereby preventing viral entry [8,33]. HBD3 binds either the HSV receptor or the HSV glycoprotein, thereby eliciting a stronger inhibition of viral entry. Furthermore, treatment of infected cells with defensins post-infection results in substantial reduction in viral replication, indicating that these peptides can exhibit post entry antiviral effects [33]. In addition, studies exploring HNP-1-3 have demonstrated their ability to reduce intracellular HSV protein transport and expression during infection [34]. HNP-1 (100 μg/mL) exhibits the greatest antiviral potential against HSV, reducing HSV titers up to 100,000-fold upon a combination of pre- and post-treatment of infected cells as compared to HNP-2 and HNP-3 (100 μg/mL), which can reduce titers by up to 100-fold following treatment [33].

#### *2.7. Respiratory Syncytial Virus*

The antiviral effects of human defensins against RSV are relatively unexplored. A study assessing leukotriene B4 (LTB4) stimulation of nasal neutrophil activity highlighted α-defensins as a possible source of antiviral activity against RSV [35]. Cells pre-treated with HBD-2 (4 μg/mL), can reduce RSV viral titers 100-fold following infection [36]. Electron microscopy images revealed damage to the lipid envelope of RSV following HBD-2 treatment, suggesting that defensins destabilize RSV virion envelopes, thereby inhibiting viral cellular entry [36]. The use of defensins in anti-RSV therapies may also limit viral evolutionary strategies that counters antiviral activities, due to the difficulty of changing the viral envelope lipid composition [37]. The evolutionary longevity of defensins suggests this to be a favorable strategy, making defensins an attractive therapeutic candidate for the treatment of RSV infections [16].
