Human metapneumovirus (HMPV), a recently discovered paramyxovirus, infects nearly 100% of the world population and causes severe respiratory disease in infants, the elderly, and immunocompromised patients. We previously showed that HMPV binds heparan sulfate proteoglycans (HSPGs) and that HMPV binding requires only the viral fusion (F) protein. To characterize the features of this interaction critical for HMPV binding and the role of this interaction in infection in relevant models, we utilized sulfated polysaccharides, heparan sulfate mimetics, and occluding compounds. Iota-carrageenan demonstrated potent anti-HMPV activity by inhibiting binding to lung cells mediated by the F protein. Furthermore, analysis of a minilibrary of variably sulfated derivatives of Escherichia coli K5 polysaccharide mimicking the HS structure revealed that the highly O-sulfated K5 polysaccharides inhibited HMPV infection, identifying a potential feature of HS critical for HMPV binding. The peptide dendrimer SB105-A10, which binds HS, reduced binding and infection in an F-dependent manner, suggesting that occlusion of HS at the target cell surface is sufficient to prevent infection. HMPV infection was also inhibited by these compounds during apical infection of polarized airway tissues, suggesting that these interactions take place during HMPV infection in a physiologically relevant model. These results reveal key features of the interaction between HMPV and HS, supporting the hypothesis that apical HS in the airway serves as a binding factor during infection, and HS modulating compounds may serve as a platform for potential antiviral development. IMPORTANCE Human metapneumovirus (HMPV) is a paramyxovirus that causes respiratory disease worldwide. It has been previously shown that HMPV requires binding to heparan sulfate on the surfaces of target cells for attachment and infection.In this study, we characterize the key features of this binding interaction using heparan sulfate mimetics, identify an important sulfate modification, and demonstrate that these interactions occur at the apical surface of polarized airway tissues. These findings provide insights into the initial binding step of HMPV infection that has potential for antiviral development.A cute viral respiratory tract infection is the most frequently observed illness in humans worldwide (1). Human metapneumovirus (HMPV), an enveloped, negative-sense, singlestranded RNA virus in the Paramyxoviridae family, is a common cause of both upper and lower respiratory tract infections (2-4). First identified in 2001 in the Netherlands, HMPV is now known to be the cause of respiratory infections in humans since at least 1958 (2). Nearly every person is exposed to HMPV in the first decade of life; seroconversion occurs on average by the age of 5 years, and nearly 100% of individuals test seropositive for antibody reactivity to HMPV antigens by the age of 10 (5). In children, HMPV infection is the second most common cause of hospitalization due to respiratory infection after the ...

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First identified in 2001, the paramyxovirus human metapneumovirus (HMPV) is a novel pathogen that causes viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide and is the second most common cause of pediatric lower respiratory illness, following the closely related RSV. Despite its clinical significance, little is known about its entry pathway, complicating the search for antivirals. Of the three surface glycoproteins expressed on the viral membrane, the attachment protein, a small hydrophobic protein, and the fusion protein F, only the F protein is required for membrane fusion and infectivity. The F protein undergoes a dramatic conformational change in order to bring the viral and target cell membranes together. This conformation change in HMPV strain CAN97–83 has been shown to be triggered by low pH. However, low pH triggering varies between F proteins of different strains. In this study we characterized fusogenic activity of strains representative of all four clades using two independent fusion assays, syncytia formation and luciferase reporter gene assay. Our results suggest that both the overall fusion level and the amount of stimulation by low pH vary between F proteins of different strains. Comparison of F protein sequence will be used to identify potential critical residues. This research was supported by NIH grant R01AI051517.

Desmogleins (Dsgs) are cadherin family members present in desmosomes and essential for epidermal integrity. Dsgs associate with lipid raft membrane microdomains, but the physiological significance of this association is not clear. Here, we report that the Dsg transmembrane domain (TMD) is the primary determinant of lipid raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. Cell based experiments revealed that the Dsg1 TMD mutant is defective in both lipid raft and desmosome association. Interestingly, molecular modeling indicates that this mutation shortens the length of the Dsg1 TMD. In addition, cryo-electron tomography of normal epithelial tissue indicates that the lipid bilayer within the desmosome is w10% thicker than adjacent regions of the plasma membrane, suggesting that hydrophobic matching of the Dsg TMD to lipid bilayer thickness contributes to the formation of a specialized membrane domain. This observation represents the first demonstration of a thicker lipid bilayer in a plasma membrane domain known to be enriched in lipid raft proteins, thereby confirming predictions about raft domains based on model membranes. Together, these findings demonstrate that differences in lipid bilayer thickness and features of cadherin TMDs drive the organization of adhesion molecules within the keratinocyte plasma membrane. Further, these findings suggest that the desmosome is a mesoscale lipid raft-like membrane domain and that mutations in Dsgs that alter raft targeting cause epidermal disease. 335Development of a new three-dimensional reconstructed human epidermis model silenced for key circadian rhythm genes to study their roles on epidermal homeostasis and 3 Biospectrum, Gyeonggi-do, Korea (the Democratic People's Republic of) Many physiological functions are under the control of a central biological clock regulated by the circadian rhythm, which follows the successive night and day cycles. At a molecular level the circadian clock consists of an auto-regulatory gene expression feedback loop driven by oscillating expression of a family of transcription factors called clock genes (Clock, Bmal, Per, Cry). The skin is the first barrier of the body against environmental aggressions such as UVs, pollution, but is also submitted to internal cellular stresses that varies a lot depending on the time of the day, therefore it is consistent that skin functions and mechanisms are monitored by circadian clock.The behavior of epidermal keratinocytes has been shown to follow a circadian rhythm, nevertheless the link between circadian rhythmicity alteration and skin integrity is largely unexplored. Besides, some recent evidences, in mice, have linked circadian rhythm abnormalities (Bmal1 KO) and skin aging. Unfortunately, the role of period genes and skin physiology and in particular skin aging was not investigated. For this purpose, we performed keratinocytes knocked down for PER2, PER3 or both expressions using sm...