Human Metapneumovirus (HMPV) Binding and Infection Are Mediated by Interactions between the HMPV Fusion Protein and Heparan Sulfate

Abstract

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 ...

Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

“…Consistent with this, recombinant hMPVs carrying these mutations replicated as efficiently as wild- type hMPV in cell culture and in cotton rats. Previously, it was shown that virus with the D331A mutation retains approximately 80% of fusion activity (25), which was consistent with our observation. In addition, we found that rhMPV-D331A was defective in viral replication in cell culture and cotton rats.…”

“…Recently, Chang et al (2012) suggested that heparan sulfate proteoglycans may function as the primary receptor for hMPV F protein (25). Mutant CHO cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), particularly heparan sulfate proteoglycans, were resistant to hMPV binding and infection, although wild-type CHO-K1 cells were permissive.…”

“…In addition, hMPV infection was abolished when CHO-K1 cells were treated with heparinases to remove heparan sulfate proteoglycans from the cell surface. Thus, it was proposed that the interaction between integrins and hMPV occurs after the initial binding of hMPV F to heparan sulfate proteoglycans (25). Perhaps the simplest explanation is that hMPV F protein uses integrins and heparan sulfate proteoglycans as coreceptors.…”

“…Furthermore, the same group of investigators showed that hMPV binds to RGD-binding integrins and that this interaction is necessary for virus attachment, viral RNA transcription, and subsequent productive infection (24). Recently, Chang et al (2012) proposed that heparan sulfate proteoglycans function as the primary receptor for hMPV and that binding to heparan sulfate proteoglycans is mediated by the F protein (25). They hypothesized that the interaction between integrins and hMPV may occur after the initial binding of hMPV F protein to heparan sulfate.…”

Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

“…For viruses in the Paramyxovirinae subfamily, it is firmly established that membrane fusion requires a specific interaction between two glycoproteins, the attachment protein (HN, H, or G) and the fusion (F) protein (1,9,14,15,23,39), and such fusion occurs at neutral pH (4,27). Specifically, it is thought that the binding of the attachment protein to cell surface receptors triggers major conformational changes in F, which in turn activates membrane fusion (1,27,29,30,39).However, membrane fusion of pneumoviruses appears to be unique among the paramyxoviruses, in that fusion is accomplished by the F protein alone without help from the attachment glycoprotein (6,7,17,21,42,43). This suggests that the F proteins of pneumoviruses possess dual functions, receptor binding and fusion promotion.…”