The paramyxovirus family contains established human pathogens such as the measles virus and human respiratory syncytial virus, as well as emerging pathogens including the Hendra and Nipah viruses and the recently identified human metapneumovirus. Two major envelope glycoproteins, the attachment protein and the fusion protein, promote the processes of viral attachment and virus‐cell membrane fusion required for entry. Although common mechanisms of fusion protein proteolytic activation and the mechanism of membrane fusion promotion have been shown in recent years, considerable diversity exists in the family relating to receptor binding and the potential mechanisms of fusion triggering.

bHuman metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), specifically, heparan sulfate proteoglycans (HSPGs), were resistant to binding and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and small hydrophobic (SH) proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding to cells did not require ␤1 integrin expression, and RGD-mediated interactions were not essential in promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking ␤1 integrin, however, were less permissive to HMPV infection, indicating that while ␤1 integrins play an important role in promoting HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F to heparan sulfate proteoglycans. H uman metapneumovirus (HMPV) is a major worldwide respiratory pathogen first isolated in 2001 from children with respiratory syncytial virus (RSV)-like infection symptoms (67).Several studies have since confirmed the importance of HMPV, generally placing it as the second or third most common cause of severe acute upper and lower respiratory tract disease in children. Though children and infants, the elderly, people with underlying cardiopulmonary conditions, and immunocompromised individuals are more susceptible to severe disease from this virus, HMPV affects people in all age groups (reviewed in reference 45). Seroprevalence studies have shown that most individuals have been exposed to this virus by the age of 5 years, though reinfections with this virus are frequent (67). HMPV infection results in a range of disease severities from mild cold-like symptoms to bronchiolitis, pneumonia, and febrile seizures and can potentially lead to death (28,45).Most paramyxoviruses express two major surface glycoproteins: an attachment protein and a fusion (F) protein. Some paramyxoviruses, including HMPV, express an additional putative membrane-spanning protein: the small hydrophobic (SH) protein (33). For a paramyxovirus to infect a cell, the virus must attach to a cellular receptor, usually through the attachment protein, and then fuse the viral and cellular membranes, a process driven by the F protein (33). Paramyxovirus F proteins are synthesized as a precursor (F 0 ) form which is then proteolytically cleaved to the fusogenically active F 1 -F 2 form (33). For HMPV, this ...

Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem-loop of the 3 ′ end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem-loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop-loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.

Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection. IMPORTANCE Human metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified. Human metapneumovirus (HMPV) is an enveloped virus belonging to the Pneumovirinae genus of the Paramyxoviridae family. HMPV is associated worldwide with severe respiratory disease, including bronchiolitis and pneumonia (1). Respiratory tract infections caused by HMPV are an important cause of hospitalizations for children under the age of five, with an annual rate of hospitalization similar to that of influenza (2). HMPV is also an important cause of severe respiratory illness in the elderly (3, 4). Studies indicate that the majority of individuals over the age of five are seropositive for HMPV (1, 5). HMPV was identified in 2001 from samples of patients with respiratory syncytial virus (RSV)-like symptoms, as symptoms of HMPV closely resemble those of RSV (5).HMPV, like other paramyxoviruses, encodes two surface glyc...