Mucins and Respiratory Virus Infection

Abstract

The airway mucus barrier is both the first point of contact between respiratory viruses and the host and a front-line defense system that viruses must overcome prior to infection. Mucin glycoproteins within this barrier protect the underlying epithelium through trapping and clearance mechanisms and influence multiple aspects of viral infection including inflammation and transmission. Further, changes in mucin expression either associated with pre-existing lung disease or induced during infection can affect disease severity. Research over the past century has aided our understanding of secreted and tethered mucin-mediated defenses, and revealed insights into specific virus-mucin interactions and the impact of both physical virion attributes and viral glycoprotein function in facilitating mucus penetration. Here we discuss these advancements and highlight efforts to apply current knowledge to the development of mucin-inspired antiviral therapeutics. Still, given the diversity among respiratory viruses and complexity of mucin biology, open questions remain, indicating avenues for continued investigation.

INVESTIGATION INTO VIRUS-MUCIN INTERACTIONS: A BRIEF HISTORY

The term “mucin” – first coined by Nicolas Theodore de Saussure in 1835 (1) – pre-dates the discovery of viruses by over 50 years. Still, even as the first human viruses were being identified in the early 1900s, little remained known about the molecular details of mucins outside of their basic chemical composition and general role in protecting epithelial surfaces. Indeed, in 1902, William Stuart-Low noted in The Lancet that: “No substance in the animal economy has been more despised and neglected of medical men than mucin, but it may yet be destined to become the head of the corner” (2).

Investigations into the impact of mucosal secretions on viral infection have now been carried out for over a century – beginning with experiments assessing the ability of human nasal washes to inhibit polio virus infection (3). Additional work in the 1940s and ‘50s, inspired by studies suggesting that mucin actually lowered resistance to bacterial infections in laboratory animals, sought to determine if mucin could similarly enhance infection by viruses in rodent models. Interest in virus-mucin interactions further increased around this time following the discovery that mucins could inhibit influenza virus-mediated haemagglutination of erythrocytes (4, 5). Moreover, the enzymatic action associated with the viral particle (later attributed to the neuraminidase (NA) (6)) not only destroyed this inhibitory capacity (4) but also resulted in the release of a “carbohydrate-peptide complex” (7) that was subsequently identified as sialic acid (8-10). Collectively, these early studies highlighted the affinity of influenza (and related viruses) for mucins and inspired their initial classification as myxoviruses (from the Greek myxa, meaning “mucus”) (11).

In the decades that followed, advancements in tissue culture opened new avenues for studying viruses in vitro, and for characterizing mucus production. This work, together with ongoing investigation of the inhibitory properties of respiratory secretions and viral NA activity, increased understanding of mucus as a barrier to infection. Meanwhile, additional studies began to characterize the impacts of viral infection on mucociliary clearance (MCC). Still, the heterogeneity intrinsic to tissue explants as well as the reliance upon histological stains and measurement of glycans or incorporation of radiolabeled sugars as tools to assess mucin expression, posed challenges. In this regard, the culture of differentiated airway epithelial cells at air-liquid interface (12, 13) that were later confirmed to recapitulate mucin expression in the human airway (14), and proven to be susceptible to a variety of respiratory viruses, was transformative. Similarly, the isolation of mucin proteins, development of mucin-specific antibodies, and successful cloning of mucin sequences, enabled improved methods for mucin detection and quantification, paving the way for more detailed studies of virus-mucin interactions in the respiratory tract. These studies have since revealed the impact of mucins on multiple stages of the viral lifecycle and identified unique viral strategies to overcome the mucus barrier, as well as opportunities to ameliorate respiratory viral infection by targeting or mimicking this critical host defense system (Fig. 1).

Fig. 1. Key aspects of virus-mucin interactions.

(1) Initial trapping: Inhaled virions are embedded within airway mucus which limits virus access to underlying epithelial cells. (2) Virus removal: metachronal cilia beating drives the transport of trapped virions up and out of the airways. (3) Immunomodulatory activity: Some virions reach the underlying epithelial cells, where membrane-bound mucins block receptor access and modulate the immune response through different mechanisms. (4) Mucin mimetic intervention: An engineered mucin mimetic can associate with viral particles, much like native mucins, illustrating the therapeutic potential of synthetic glycopolymers reinforcing mucosal defenses. (5) Transmission: Mucin proteins associate with shed viral particles in respiratory droplets expectorated from infected lungs. Created in BioRender. Scull, M. (2026) https://BioRender.com/yw0x27g

AIRWAY MUCINS AND THE MUCUS BARRIER

Organization of the mucus barrier and mucin expression

The airway mucus barrier sits at the interface between the external environment and underlying epithelial cells and is composed of two layers: the secreted, mobile mucus gel and underlying periciliary layer (PCL; Fig. 2A.). The mobile mucus is a non-Newtonian fluid composed of proteins, lipids, ions, water, cell debris, and secreted mucins, while membrane-bound (“tethered”) mucins populate the PCL. Examination of these two layers by electron microscopy (15, 16) and histology (17) shows a clear distinction between the mucus gel, which is approximately 10–100 μm thick, and the PCL, which extends 7–10 μm from the apical cell surface, similar to the height of cilia (18). Together, these observations have given rise to the current “gel on brush” model (18), where the net negative charge associated with the tethered mucins attracts water and promotes inter-mucin repulsion. As a result, the PCL has a higher osmotic modulus than the mobile mucus gel (19), preventing gel-forming mucins from penetrating the interciliary spaces while maintaining the association between the secreted mucus layer and the cilia. Cilia beating within the hydrated PCL propels the mucus gel out of the airways, ensuring efficient MCC.

Fig. 2. Mucin expression and anti-viral functions in the respiratory mucosa.

(A) ”Gel-on-brush” model of the airway mucus barrier between the external environment and epithelial cells. The mucus layer forms a mesh made of MUC5AC (red) and MUC5B (blue) which are decorated with glycans containing different terminal residues, such as sialic acid (SA; see legend) and secreted via mucus plumes from goblet cells. Incoming viral pathogens from the external environment, such as IAV (spherical (S); filamentous (F)), adenovirus, human metapneumovirus (hMPV), RSV (spherical (S); filamentous (F)), CoV, RV, hPIV, and adeno-associated virus (AAV), get trapped or are hindered by the mucin-rich mucus layer. Some of these viruses utilize SA-binding and –solubilizing mechanisms to penetrate the mucus layer, leaving behind mucins that lack the terminal SA. Differences in particle shape and distribution of surface HA and NA on IAV (S) and IAV (F) impact virus mobility in mucus (indicated by long vs. short dashes, see legend). Viruses that overcome the secreted mucus gel reach the PCL and interact with mucins such as MUC1, MUC4, and MUC16. Larger viruses such as adenovirus, IAV, and SARS-CoV-2 may be excluded from the PCL based on size, while smaller viruses like AAV can pass through unhindered. RSV and SARS-CoV-2 can trigger “podia-like” projections or microvilli extensions, respectively, on the epithelial cell surface, which can facilitate infection and spread. (B) Cross section of the airway mucus barrier and epithelial cells depicting the immunomodulatory effects of mucins during viral infection. 1. The secreted mucus layer houses soluble antiviral compounds that affect incoming viral pathogens and IgA/IgG antibodies that can trap viruses (e.g., IAV). It is also the site where secreted MUC5B and tethered MUC4 can trigger eosinophil apoptosis following interaction with Siglec-F (as described in mice). 2. MUC1 expression on infected epithelial cells (indicated by transparent viruses) is increased during IAV and RSV infection and may modulate the immune response via its cytoplasmic tail (see main text for details). IAV induced cytokines also lead to increased MUC1 expression on macrophages, while MUC5AC upregulation during RV infection is associated with elevated cytokine levels and neutrophil influx. 3. In addition to epithelial cells and macrophages, MUC1 is also expressed on the surface of dendritic cells and T-cells, where it may influence activation state and proliferative capacity. Created in BioRender. Scull, M. (2026) https://BioRender.com/0zavl2q

The major mucin glycoprotein components of the highly viscoelastic secreted mucus are mucin (MUC) 5B and MUC5AC, where MUC5B is the main mucin protein found in the gel forming layer in healthy airways (20). MUC5B is produced by submucosal glands throughout the proximal and distal airways as well as by some goblet cells (21), while MUC5AC is the predominant mucin secreted by goblet cells in the surface epithelium, located in the proximal cartilaginous airways (21, 22). Though at lower quantities than MUC5B, the presence of MUC5AC in the distal airways may be explained by the extension of goblet cells into the gland ducts (21, 22). This regional variation in mucin expression throughout the lung underlines the functional complexity of the mucus barrier.

MUC1, MUC4, and MUC16 are the predominantly expressed mucins within the airway PCL, with others such as MUC13, MUC15, MUC20, MUC21, and MUC22 at markedly lower levels (23). Within the PCL, immunohistochemical analysis localizes MUC1 to microvilli and MUC4 to cilia, while MUC16 is preferentially expressed on the apical membrane of goblet cells (24). Notably, although these mucins are classified as “tethered,” they are also frequently detected in the gel forming layer, which is likely due – at least in part – to the fact they can be shed from the cell surface or incorporated into extracellular vesicles (25).

Physical structure of mucins

The structural characteristics of mucins are important as they give rise to their defensive properties and enable virus interactions within mucus barriers. The structures of the major transmembrane and secreted mucins have been previously reviewed in detail (23, 26). In short, most major airway mucin proteins are characterized by an extensive core domain comprising a variable number of tandem repeats (VNTRs) that are rich in proline, threonine, and serine residues (27, 28). These residues are sites for extensive glycosylation; in fact, nearly 80% of the resulting polypeptide mass is attributed to these carbohydrate chains (29). The high density of O-glycans produces a bottlebrush-like structure, with a peptide backbone held rigid by the steric hindrance and repulsion of the glycan moieties, resulting in a more linear conformation than that of conventional proteins (29, 30). The extensive glycosylation that occurs also makes mucin proteins highly heterogeneous, leading to diversity in the overall size and charge of similar mucins. In addition, distinct MUC5B glycoforms have been identified within specific regions of the airways, suggesting local cell populations may produce unique glycoforms due to cell-specific patterns of glycosyltransferase expression (20). Structural variation of mucin proteins also results from differences in their VNTR regions and isoform generation. Many mucin proteins with long VNTRs are highly polymorphic in repeat numbers which can significantly alter the protein’s molecular weight (31). In some cases, these alterations have been linked to chronic lung disease outcomes, suggesting these changes can influence the functional properties of the protein (31). Similarly, alternative spicing can generate truncated isoforms of particular mucins, impacting their size, glycan profile, and role in cell signaling in the case of transmembrane mucins (32).

The tethered mucins, MUC1, MUC4, and MUC16, range in size from 250kDa (MUC1), to >2MDa (MUC16) and are characterized by three principal domains: the densely O-glycosylated extracellular domain, transmembrane domain, and short cytoplasmic tail (33). Following synthesis, the tethered mucin peptides generally undergo autoproteolytic cleavage, resulting in two subunits that remain tightly associated at the cell surface (34). While the extracellular domain participates in the barrier function of the glycocalyx as well as maintaining hydration within the PCL, the cytoplasmic tail of each mucin is well-conserved across species and may be involved in cell signaling (35). For example, the cytoplasmic tail of MUC1 has over 20 predicted phosphorylation sites, many of which have been experimentally validated and shown to be involved in modulating cell growth and survival via interaction with host factors such as β-catenin (33).

The secreted, gel-forming mucins of the mucus barrier (MUC5B and MUC5AC), are similar in size (>2MDa) and domain architecture, but have distinct tandem repeat patterns and key differences within their N-terminal regions (26). These differences impact disulfide bond formation between mucin monomers, where MUC5AC undergoes more extensive, higher-order oligomerization giving rise to net-like multimers compared to the mainly linear dimers formed by MUC5B (36, 37). These different behaviors of MUC5B and MUC5AC confer unique architectures and biophysical properties of the mucus gel.

MUCIN-MEDIATED DEFENSES AND RESPIRATORY VIRUS COUNTERMEASURES

Virus trapping through adhesive interactions

Extensive glycosylation is clearly a defining feature of mucins, and the carbohydrate chains that decorate the mucin protein core often terminate with blood-group (Lewis) epitopes, sialic acid (Sia), fucose, or sulfate residues (38). Notably, many viral glycoproteins recognize complex host cell glycans such as Sia and heparan sulfate proteoglycans (HS), and these interactions are often essential for target cell attachment and entry. Therefore, mucin-associated glycans can act as “decoy receptors” during viral infection (39). Influenza A virus (IAV), for example, binds Sia via its haemagglutinin (HA) protein and, even though this interaction is weak and reversible (40), multiple studies suggest Sia-mediated trapping occurs (Fig. 2A). For example, one group described an inverse correlation between the Sia content of human salivary mucins and infection efficiency in vitro (41), while another demonstrated that the antiviral properties of bovine submaxillary mucins (BSM) towards IAV H3N2 subtypes were reduced 10-fold upon removal of Sia (42). Similarly, Ehre et al. reported reduced IAV viral loads in the lungs of human Muc5AC transgenic (Tg) mice, and further linked this to the α2,3-Sia on Muc5AC present in the bronchoalveolar fluid (43). Notably, sialoglycomes differ between species and this may affect virus mucus penetration and the zoonotic transmission potential of viruses, like IAV, that bind Sia (44). IAV was also shown to bind MUC1 – or presumably MUC1-associated glycans – on cell lines, leading to the hypothesis that MUC1 acts as a releasable decoy whereby shedding of the virus-mucin complex from the cell surface limits further infection (45). Consistent with this model, our group also found that IAV could interact with endogenous MUC1 in human airway epithelial (HAE) cultures, although MUC1 shedding was not observed (46). Nonetheless, enhanced viral replication and spread – even for mutant IAV viruses with altered sialic acid binding preference – in MUC1-depleted HAE models supports a barrier function of MUC1 against endocytic viral uptake (46).

Glycan binding also plays a central role in infection by other human respiratory viruses, including multiple coronaviruses (CoVs). Similar to influenza C virus, HCoV-OC43 (47) and HCoV-HKU1 (48) bind to 9-O-acetyl-sialic acid as a primary receptor, while α2,3-linked sialic acid has been proposed as a co-receptor for Middle East Respiratory Syndrome (MERS)-CoV (49). Additional interactions between HCoV-NL63 and HS (50), and Severe Acute Respiratory Syndrome (SARS)-CoV-2 and HS (51), Sia (52), and even type I blood group A antigen have been reported and suggested to promote infection (53). This indicates that adhesive interactions between mucins and respiratory viruses are not limited to influenza. In support of this, Wang et al. demonstrated direct binding between MUC1 and SARS-CoV-2 and further suggested, based on molecular dynamics simulations, that the observed progressive decline in affinity for MUC1 across wild type, Delta, and Omicron Spike proteins indicates that more recent variants are better poised to overcome MUC1 defenses (54). However, glycans were not considered in these experiments, and additional studies showing direct binding of CoVs to native human mucins are lacking. Nonetheless, interaction between HCoV-OC43 and sialic acids present on bovine mucins was demonstrated using an ELISA-inspired assay incorporating BSM in its native, denatured, and desialylated forms (55). Further, bovine mucins effectively blocked HCoV-OC43 infection in a glycan-dependent manner and only when the Neu5Ac was present on the mucin backbone and not in soluble form (55). Additionally, mathematical thermodynamic equilibrium modeling of MERS-CoV-mucin interaction supports the idea that mucins trap the virus and impede it from reaching target cells (56).

The ability of viruses to overcome glycan-mediated interactions with mucins is largely attributed to the enzymatic activity associated with viral glycoproteins such as the IAV NA (Fig. 2A). Although first implicated in the release of viral progeny from infected cells (57), later work by Matrosovich et al. provided experimental evidence in support of an additional role for IAV NA during initial infection (58). This possibility gained further traction through studies showing that NA promotes swine influenza virus diffusion in sialic acid rich mucus (59) and that sialylated mucins present in human mucus are, in fact, cleaved by NA in the early stages of IAV infection (41). NA-directed movement and proper balance of the respective sialic acid-binding and -solubilizing actions of IAV HA and NA are now seen as essential for efficient mucus penetration and the “crawling” and “gliding” movement of IAV along the cell surface (60). Notably, IAV NA activity is strain-dependent and inversely correlates with virus sensitivity to mucus (61), while longer NA stalk length (as seen in human-adapted IAV) has also been proposed to facilitate mucus penetration (62). Navigation through mucus by other respiratory viruses, such as Newcastle disease virus, Sendai virus, and human parainfluenza virus 3 (hPIV3), was also shown to be dependent on their sialic acid receptor-binding and -cleaving activities (63). All three viruses bind to α2-3-linked Sia receptors along with hPIV3, which also interacts with α2-6-linked Sia, followed by sialidase-driven cleavage of sialoglycans and virus release. This imparts a “burnt-bridge ratchet / lawnmower-like” movement of viral particles through mucus (63). Similarly, influenza C virus and 9-O-acetyl-sialic acid-binding CoVs harbor receptor-destroying enzyme activity in their haemagglutinin-esterase (HE) protein (64). The viral HE can reversibly bind to BSM and release the acetyl group from N-acetyl-9-O-acetylneuraminic acid, which is hypothesized to facilitate migration of the virus through the mucus layer and later promote viral release and spread throughout the epithelium (65).

Importantly, viral particles can also adhere to mucins through electrostatic interactions, and these interactions likely vary between non-enveloped and enveloped viruses as a result of differences in virion composition that influence surface chemistry (39, 66). Viral capsid structures that have a net-neutral charge and lack external hydrophobic regions that could interact with mucin domains involved in the formation of mucin networks, appear particularly well-suited for mucus penetration (39). This is exemplified by the ability of Norwalk and human papillomavirus-like particles to easily traverse cervical mucus (67), though further studies examining the effects of surface charge associated with specific respiratory viruses on their passage through airway mucus are warranted.

Steric hindrance of viral particle penetration

Independent of adhesive interactions, the nanoscale porous network formed by secreted mucin oligomerization and physical entanglements can sterically block viral particle penetration (Fig. 2A). Indeed, although the ~500 nm pore size of the secreted mucus mesh is large enough to accommodate most viral particles, a recent study from our group highlights the importance of the pore structure and crosslinking of mucus gels in IAV mobility (40). Further, the contribution of specific secreted mucins like MUC5AC to steric hindrance of viral particle penetration was demonstrated in Muc5AC-deficient mice during respiratory syncytial virus (RSV) infection, where a lack of Muc5AC resulted in a more porous barrier and loss of airway protection, leading to increased viral replication and acute lung injury (68). Similarly, tethered mucins, which extend up to 1500 nm from the cell surface and boast dense glycosylation, constitute a physical barrier towards particles – especially those >40 nm in diameter – entering the PCL (18, 24). In accord, the PCL has been shown to exclude adenoviruses (100 nm) while allowing adeno-associated viruses (30 nm) partial penetration before trapping (24). These observations have established the mucus barrier as a molecular sieve, and have led to the idea that the small sizes of viruses were favored through natural selection, conferring an advantage in penetrating the glycocalyx (39).

The contribution of specific mucins to the PCL barrier is highlighted by studies in which depletion of tethered mucins boosts viral infection – potentially by increasing PCL permeability, or by exposing underlying receptors. Studies using synthetic mucin mimics, for example, showed that when tethered mucins are at high density, they undergo conformational extension and sterically hinder IAV from accessing underlying glycoconjugates (69, 70). Likewise, enhanced uptake of SARS-CoV-2 or Spike-bearing pseudoparticles following enzymatic removal of the MUC1 extracellular domain (MUC1-ED) from Calu-3 cells and primary human airway organoids was attributed to the proximity of MUC1-ED and ACE2 on the Calu-3 cell surface and the likelihood that MUC1-ED was blocking virus-ACE2 interaction (71). Additional evidence that MUC1 inhibits viral access to underlying airway epithelial cells is provided by studies in knockout mice where the absence of Muc1 yielded an increase in IAV and mouse adenovirus type 1 viral titers, as well as an increase in adenoviral vector-mediated gene transfer efficiency compared to wild-type animals (45, 72, 73). In addition to MUC1, the extracellular domains of MUC4, MUC13, and MUC21 have also been shown to restrict SARS-CoV-2 by blocking virus binding and entry, with conserved effects across SARS-CoV-2 variants and other CoVs (74, 75).

Beyond viral particle size, current evidence suggests particle morphology also influences mucus penetration. Respiratory viruses such as influenza and RSV are pleomorphic, with filamentous forms measuring up to several microns in length (76, 77). For IAV, the production of filamentous particles, which are observed in natural infections and lost during passage in cell culture, is suggested to be a viral adaptation to the mucosal microenvironment (78). Although this seems counterintuitive, given that filamentous particles are larger than their spherical counterparts (76) and have more plausible multivalent HA-Sia interactions (40), prior work has shown that rod-shaped nanoparticles are actually more efficient at penetrating mucus barriers due to rotational dynamics (79). Furthermore, recent work demonstrates that IAV filaments display asymmetric distribution of HA and NA on their membrane (80), which promotes persistent movement away from their NA-rich pole (81).

Viruses may also take advantage of the mucociliary machinery to penetrate the dense PCL. This concept was recently explored by Wu et al. who proposed that SARS-CoV-2 binds to ciliary ACE2 receptors, leading to fusion at the cilium surface, or trafficking of the virus-ACE2 complex along the cilium to the cell body prior to fusion (82). Notably, depletion of motile cilia by CEP83 knockdown had no significant impact on viral receptor expression but reduced infection by SARS-CoV-2, RSV, and hPIV3, further indicating that cilia provide a conduit through the PCL that is broadly utilized across respiratory viruses (82). Moreover, RSV attachment at the cell surface was shown to trigger the formation of “podia-like” projections that penetrate the PCL; expression of viral receptors on these projections, and the proximity of these extensions to viruses still attached to cilia suggest an additional mechanism by which RSV overcomes the glycocalyx (83). Later in infection, SARS-CoV-2 appeared to trigger microvilli extensions, promoting viral egress into the mucus layer and subsequent spread within the mucosal environment (82).

Removal of viral particles through mucociliary clearance

The defensive properties of the mucus barrier towards respiratory virus infection are ultimately dependent on the continuous clearance and renewal of the barrier itself. Clearance results from the complex interplay between the secreted mucus and PCL, which, when paired with the metachronal movement of cilia, creates a “mucociliary escalator” that acts to remove inhaled viral particles from the respiratory tract. MUC5B and MUC5AC contribute to mobilization and directional transport of mucus, respectively (84), while tethered mucins promote MCC by facilitating ciliary beating (24).

The rate of MCC – on the order of 10-100 μm per second – and site of viral particle deposition along the respiratory tract critically influence how quickly a virus must penetrate the mucus barrier before it is removed to the gastrointestinal tract for neutralization (Fig. 3). Not surprisingly, respiratory viral infections have been linked to altered cilia beat frequency, disordered or remodeled cilia, as well as loss of cilia structures or ciliated cells in their entirety through a variety of mechanisms that would abrogate effective MCC over the course of the infection (85). Accumulation of cell debris or mucus overproduction as observed during SARS-CoV-2 (66) and RSV (86) infections can also influence MCC, as discussed below. Still, experiments using agarose overlays or knock-out of ciliary dynein genes to abrogate MCC in vitro, suggest that MCC may actually benefit some respiratory viruses, such as SARS-CoV-2, by promoting spread, at least during the initial phase of infection (87). These data are in alignment with clinical reports indicating that individuals with primary ciliary dyskinesia (who lack functional MCC), are not at greater risk for severe COVID-19 (88). Similarly, the spread of RSV to nearby cells during infection of airway epithelial cultures has also been linked to ciliary movement and mucus transport (89).

Fig. 3. Comparison of healthy and diseased airways during initial and acute phases of infection.

(A) Healthy airway. Initial infection: A healthy epithelium features a MUC5B-rich mucus barrier and hydrated PCL promoting cilia beating that traps and readily clears most inhaled virions by MCC, although a small number of virions navigate the barrier and initiate infection. Acute infection: As infection progresses, immune responses cause an increase in mucus secretion and changes to mucin protein expression, namely increased MUC5B and significantly increased MUC5AC expression. MCC slows slightly over time, though a considerable degree of clearance persists until the infection is cleared. (B) Chronic disease airway. Initial infection: The epithelium in chronic disease has a thickened, MUC5AC-rich mucus barrier at baseline prior to infection, trapping inhaled virions but allowing them more time to reach underlying target cells before clearance. Acute infection: As infection proceeds, immune responses including inflammation and increased goblet cell hyperplasia over baseline add to the already thickened mucus layer. This substantially increases mucin secretion, resulting in MUC5AC tethering, compressed cilia with ineffective beating, and significantly slowed MCC prolonging viral persistence. Created in BioRender. Scull, M. (2026) https://BioRender.com/5gcpqqu

Mucin-mediated immunomodulation

Beyond their more direct roles in virus trapping and removal, mucins also influence antiviral defense through modulation of the host response (Fig. 2B). Specifically, mucins can scaffold soluble factors, influence intracellular signaling pathways, and affect immune cell function. Lysozymes and lactoferrin, for example, which have potential anti-viral properties, have been found in association with airway mucins (90). Antibodies can also interact with mucins via their Fc region forming transient, weak and low affinity crosslinks that avidly trap antibody-coated viruses in mucus (91). In fact, it has been reported that IAV immobilization in mucus is not solely dependent on direct viral HA-Sia binding, but also on influenza-specific IgG- and IgA-mediated restraint of viral particles present in the mucus (92). IAV filaments may also function to sequester IgA antibodies present in the mucus layer and act as immune decoys (93).

MUC5B and MUC5AC can also impact inflammation, although assessing these impacts during viral replication is often complicated by the fact that manipulation of MUC5B/AC expression can alter host susceptibility to infection (43, 68). Nonetheless, MUC5AC concentration in the sputum of rhinovirus (RV)-infected chronic obstructive pulmonary disease patients exhibited a positive correlation with neutrophil counts and cytokine levels (94), suggesting MUC5AC is pro-inflammatory. This conclusion was further supported by RV infection in Muc5ac^−/− mice where reduced levels of inflammation were observed compared to wild-type controls despite similar viral loads (94). Reduced cytokine expression and neutrophil influx were also observed during acute lung injury induced by mechanical ventilation in Muc5ac^−/− mice (95). Furthermore, the addition of exogenous MUC5AC in wild-type mice and HAE cultures infected with RV led to the release of extracellular ATP, providing a potential mechanism through which MUC5AC exerts pro-inflammatory effects (94). Studies in Muc5b^−/− mice also demonstrate altered inflammatory profiles, with notable effects on macrophage function (96, 97).

Tethered mucins may also have a direct impact on the host response to viral infection through cytoplasmic tail-mediated interactions with intracellular signaling pathways (98). These interactions have been most extensively studied for the cytoplasmic tail of MUC1, which affects cell survival pathways – for example, in Calu-3 cells during SARS-CoV-2 infection (99) – and acts as a universal negative regulator for multiple Toll-like receptors (TLRs), including several involved in viral recognition (100). The role of MUC1 in airway inflammation is further exemplified during RSV infection, in which tumor necrosis factor (TNF)α triggers MUC1 upregulation, which in turn suppresses TNFα release (101). MUC1 has also been suggested to regulate influenza virus-induced production of pro-inflammatory cytokines interleukin (IL)-6 and MCP-1, thereby protecting against severe pulmonary disease (45). Other membrane bound mucins such as MUC4 and MUC16 have the potential to affect signaling pathways (23), although their immunomodulatory effect during respiratory viral infection requires further investigation.

More broadly, macrophages, dendritic cells, and activated T cells, which are important components of the immune response during viral infection, also express MUC1 on their surface (33). Correspondingly, our group has shown that soluble factors produced during IAV infection cause an increase in MUC1 expression on primary human monocyte-derived macrophages; although the functional consequences of this were not tested, work by others studying bacterial pathogens suggests potential impacts on phagocytosis (46, 102). Further, cross-linking MUC1 on activated T cells inhibits proliferation and suggests an immunoregulatory function similar to other immune checkpoint molecules like PD-1 (103), while mathematical modeling indicates MUC1 expression on epithelial cells limits macrophage accumulation during influenza infection (104). Likewise, mucin-leukocyte interaction via Siglec expression on alveolar macrophages and eosinophils are posited to have a role during lung homeostasis and inflammation (105). For instance, MUC5B and MUC4 can bind to Siglec-F on murine eosinophils in a sialic acid dependent manner, triggering apoptosis (106). Collectively, these reports suggest that MUC1 and other mucins have significant potential to influence immune cell responses during respiratory viral infection.

ALTERED MUCIN EXPRESSION AND IMPLICATIONS FOR VIRAL INFECTION

Variation in mucin expression during respiratory viral infection: mechanisms and biomarkers of severe disease

Mucin expression undergoes dynamic changes during respiratory viral infection, impacting the composition and structure of mucus and altering the properties of the mucus barrier. Increases in MUC5B expression and significant increases in MUC5AC are particularly common among respiratory viral infections, though the underlying mechanisms vary between viruses (Fig. 3). For instance, the RSV fusion protein can interact with epidermal growth factor receptor (EGFR) and subsequently mediate mucin expression (107), while viruses with a dsRNA intermediate may induce mucin expression via a TLR3-EGFR dependent pathway (108, 109). RSV (110) and RV infections in early-life (111) have been shown to induce Th2 immune signaling, activating cytokines such as IL-4 and IL-13 that drive persistent mucus metaplasia, like that seen in chronic bronchitis. Like RSV, human metapneumovirus infection also results in increased mucin expression, though the expression profile of particular mucins, specifically MUC1, MUC2, and MUC5B, differs from those in RSV infection (112). In contrast, severe influenza infection has been shown to cause increased expression of IL-1 and TNFα (113), inducing MUC5AC gene expression (114, 115). Severe SARS-CoV-2 infection of HAE cultures has revealed similar roles of both EGFR and IL-1R pathways in mucin gene upregulation. The result of which is increased MUC5B expression and regional MUC5AC accumulation, with MUC5B dominating mucus plugs found in the distal airways of deceased subjects with COVID-19 lung disease (116).

Given these changes, mucin expression analyses and glycan profiles have been used to analyze and potentially predict viral disease severity. Increased MUC4 production in particular has been shown in bronchioles with necrotizing bronchiolitis during IAV infection, and an increase in MUC5AC levels in mucus from the upper airways is correlated with increased RSV disease severity (117, 118). Additionally, increases in the tethered mucins such as MUC1 are associated with more severe SARS-CoV-2 disease outcomes (119, 120), though their presence prior to initial infection has an antiviral effect (74). Genetic analyses have also identified links between severe respiratory virus infection outcomes and single nucleotide polymorphisms indirectly influencing mucin expression (121) or occurring within mucin genes themselves (122, 123).

Altered mucin expression as a function of underlying lung disease and age: impacts on susceptibility to respiratory virus infection

Mucin expression also undergoes significant alterations in chronic lung disease states. For example, in asthma, mucus has been found to be unusually solid and enriched in high molecular weight mucins, particularly a lower charge glycoform of MUC5B, altering mucus biophysical properties and contributing to the formation of obstructive mucus plugs (124, 125). Additionally, MUC5AC has been shown to exhibit epithelial tethering in asthma, impairing mucociliary transport and likely also promoting mucus plugging (126). In cystic fibrosis, submucosal glands and nasal polyps of upper nasal mucosa show increased MUC5B expression, with nasal polyps additionally exhibiting elevated levels of tethered mucins including MUC1 and MUC4 compared to healthy tissue (127). In many cases of chronic bronchitis or asthma, IL-13 is a major factor in Th2-driven inflammation, which also has been shown to elevate both MUC5B and MUC5AC via goblet cell hyperplasia (128). The result of which is a thickened mucus layer leading to impaired clearance and increased airway obstruction contributing to exacerbation of disease. Collectively, these baseline alterations in the mucus barrier may influence respiratory viral infection; indeed, higher frequencies of respiratory viral infections are observed in patients with chronic airway diseases (129, 130). However, the mechanisms behind this correlation are not well understood. On one hand, mucin specific differences in disease states, namely the increase in MUC5AC over MUC5B, results in a more viscoelastic mucus with a tighter network of mucin proteins, enhancing the barrier. On the other hand, these differences reduce clearance, allowing a trapped viral particle more time to reach target epithelial cells and potentially contributing to viral persistence in the airways. Changes in tethered mucin expression, such as the decrease in MUC1 in bronchial epithelial cells of severe asthma patients (131), may also play a role in viral pathogenesis.

Since clinical phenotypes of acute airway infection and chronic airway diseases have been shown to be age-dependent, several studies have sought to identify age-dependent variations in the expression and glycosylation patterns of both tethered and secreted mucins. A comparative study using primary nasal cultures derived from individuals of either ≤10 or ≥60 years of age revealed a higher overall expression of MUC5AC in cultures from young children, while the expression of MUC5B was not significantly different between age groups (132). Moreover, proteome analysis shows an upregulation of proteins related to glycosylation and secretion of mucins in children. These findings may provide insight into the age-dependent phenotypes of acute and chronic airway disease, as well as provide an explanation for the predisposition of younger children to allergic asthma or other muco-obstructive diseases more common in children. Additionally, the expression of the tethered mucin Muc1 in the lungs of mice has been shown to peak in early development, and gradually decrease over time, along with some of the most abundant Galnt proteins, which are involved in O-glycosylation (133). Because the expression patterns of multiple mucins and mucin-type O-glycosylation proteins change over a lifetime and generally decrease with age, the barrier function of mucus against respiratory viral infection also changes. The result of this dynamic expression, however, can be a double-edged sword, where increased mucin at younger ages better trap inhaled viral particles compared to the lessened mucus barrier in elderly airways, while increasing the chance of mucus plugs in the younger, and smaller, airways. In addition to these changes in mucus and mucin expression, mucus clearance rates are also altered with age, with significantly slower rates of mucus movement in nasal epithelium described for individuals over 40 years old (134). Both the change in mucin expression and loss of efficient MCC during aging may contribute to the disproportionate burden of respiratory infections in the elderly.

IMPACTS OF MUCINS ON VIRAL TRANSMISSION BETWEEN HOSTS

In addition to the interplay between mucins and respiratory viruses within the lung microenvironment, mucins can also affect respiratory viral particles during transit to the next host and can thereby influence transmission efficiency. Transmission is essential for respiratory virus maintenance within a population and can occur through different mechanisms (e.g., contact with infected individuals or contaminated surfaces (fomites), large droplets, and fine aerosols (135)) all of which require the virus to remain infectious in the external environment for some period of time. Since viruses are shed in combination with respiratory secretions, multiple studies have investigated the impacts of mucins or human mucus sourced from airway cultures or volunteers on virus infectivity over time, alongside temperature and humidity. These studies indicate that while mucus can promote respiratory virus survival in both aerosolized particles and on a variety of surfaces – up to 17 days for IAV in one study (136) – that the impacts can be variable depending on virus strain (136, 137), donor source of the mucus (138), and humidity level (139-141). Challenging the later, Kormuth et al. (142) studied the viability of IAV and φ6 bacteriophage in both aerosols and droplets and reported that airway surface liquid from human airway epithelial cultures protected these viruses from decay over a range of relative humidities. Still, mucus-mediated protective effects may be transient. For example, viral infectivity in aerosols was preserved when foot-and-mouth disease virus was suspended in nasal fluid (143), or when coronavirus was mixed with porcine gastric mucins (144), but only over short time scales (i.e., within the first few minutes after aerosolization).

Additional studies have taken aim at the mechanism by which mucins may impact virus stability in respiratory droplets. While current evidence suggests mucins do not alter aerosol evaporation kinetics, they do influence bioaerosol microphysics, leading to the formation of inclusions within droplets that may shield viruses from inactivation (144). In line with this, influenza virions were shown to co-localize with mucins within droplets, likely via HA-mucin interactions, suggesting that mucins could protect the virus from high solute concentrations (145). Wardala et al. also observed a physical association between HCoV-OC43 and mucins on fomites; however, this interaction appeared to block subsequent infection of cells (55). Thus, counter to their protective effects in the external environment, mucins also have the potential to neutralize particles in transit.

ENGINEERING MUCIN-LIKE MOLECULES AS ANTIVIRALS

Myriad interactions between mucins and respiratory viruses described above limit efficient infection in the lung, as such, mucin biomimetics made to replicate properties of mucins and mucus have been sought out to act as antivirals against respiratory viruses. Because glycan composition is decidedly variable among mucins and mucin-like proteins, mucin-mimetics are highly manipulable to have dynamic compositions. For example, MUC1 has been synthetically modeled and directly shown to inhibit IAV infection in vitro (45). Additionally, sialylated oligosaccharides or sialic acid analogues such as Zanamivir attached to a polymer backbone to mimic glycosylated mucins may work as antivirals against viruses with sialyl linkage specificities, like IAV (146-148). Mucin-like molecules can be engineered to change their polymer length, glycosylation, and overall charge in order to fine tune their antiviral capacities against particular viruses. Tethered mucins of the glycocalyx can similarly be modeled with lipid-tethered synthetic glycopeptides, in this case to use as a reductionist model to further investigate their roles in viral pathogenesis (69). These tethered mucin-mimetics likewise allow the control of glycan composition and length from the cell surface in the glycocalyx.

Antibodies made to tightly bind a virus can also be made to associate with mucins, effectively keeping the virus stuck in mucus until it can be dispelled from the airway (149, 150). For example, the neutralizing mAb motavizumab binding RSV F has been engineered with Fc regions that can form lower-affinity bonds with mucins, immobilizing the virus within the mucus (149). This type of antiviral can be optimized as an inhalable therapy, directly targeting virions at the mucosal surface, where systemically administered therapies have been lacking in efficacy. The potential of exploiting the native role of mucus to enhance trapping, as well as engineering mucin biomimetics to augment the antiviral functions of mucus is a promising route in developing novel antivirals.

CONCLUDING REMARKS

Respiratory virus interaction with mucins is essentially unavoidable during infection. Not only are respiratory viruses required to traverse the mucus barrier to access underlying target epithelial cells, but mucins impact multiple stages of the viral infection cycle including subsequent spread within the airways, and transmission between hosts. Through continued research, themes have emerged regarding the mechanisms that allow viruses to counter adhesive and steric interactions within the mucus barrier. Still, which mucins exert antiviral defenses against which viruses has not been fully delineated, and how viral infection is impacted by known variation in mucin structure, – including glycosylation, which may be further modified by the resident microbiota or bacterial co-infection – needs to be investigated. Additional gaps in knowledge relate to the influence of mucin expression on epithelial and immune cell interactions as well as tissue recovery. Furthermore, data identifying cases in which mucins can aid infection, potentially by promoting virus retention on the cell surface (70, 74), should be pursued. Whether viruses budding from airway epithelial cells incorporate mucins into their envelopes, and whether this impacts virus neutralization or the ability of viral particles to navigate the mucosal microenvironment also remains unexplored. Given the dynamics of the mucus barrier and multifunctional nature of individual mucins, answering these questions will require innovative approaches and integration of tools spanning the fields of virology, glycobiology, biophysics, and immunology. These efforts will yield new insights into virus-host interactions in the airways that can guide therapeutic strategies, or be applied to improve the design of engineered vectors targeting the respiratory epithelium.