Human Metapneumovirus Binding and Infection is Mediated 1 by Interactions between the HMPV Fusion Protein and 2 Heparan Sulfate 3

23 Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper 24 and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its 25 target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and 26 infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type 27 CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize 28 glycosaminoglycans (GAGs), specifically heparan sulfate proteoglycans (HSPG), were resistant to binding 29 and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells 30 were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and SH 31 proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein 32 (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding 33 to cells did not require β1 integrin expression and RGD-mediated interactions were not essential in 34 promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking β1 integrin, however, were less 35 permissive to HMPV infection, indicating that while β1 integrins play an important role in promoting 36 HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F 37 to heparan sulfate proteoglycans. 38 39 on Jauary 9, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Introduction 40 Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen first isolated in 2001 from 41 children with respiratory syncytial virus (RSV)-like symptoms (68). Several studies since have confirmed 42 the importance of HMPV, generally placing it as the second or third most common cause of severe acute 43 upper and lower respiratory tract disease in children. Though children and infants, the elderly, people 44 with underlying cardiopulmonary conditions, and immunocompromised individuals are more susceptible 45 to severe disease from this virus, HMPV affects people in all age groups (reviewed in (46)). 46 Seroprevalence studies have shown that most individuals have been exposed to this virus by the age of 47 five though reinfections with this virus are frequent (68). HMPV infection results in a range of disease 48 severities from mild cold-like symptoms to bronchiolitis, pneumonia, and febrile seizures, and can 49 potentially lead to death (29, 46). 50 Most paramyxoviruses express two major surface glycoproteins: an attachment protein and a fusion (F) 51 protein. Some paramyxoviruses, including HMPV, express an additional putative membrane-spanning 52 protein: the small hydrophobic (SH) protein (34). For a paramyxovirus to infect a cell, the virus must 53 attach to a cellular receptor, usually through the attachment protein, and then fuse the viral and cellular 54 membranes, a process driven by the F protein (34). Paramyxovirus F proteins are synthesized as a 55 precursor (F0) form which is then proteolytically cleaved to the fusogenically active F1+F2 form (34). For 56 HMPV, this cleavage is accomplished by an exogenous protease (54, 55). This proteolytic cleavage 57 primes the F protein for triggering, which, for some clades of HMPV, is driven by low pH (28, 54). There 58 is no evidence that the SH protein plays a role in viral entry. In fact, HMPV SH protein is dispensable for 59 virus growth in vitro and in vivo (4). 60 The paramyxovirus attachment protein is a type II integral membrane protein called either HN, H, or G 61 (34). Paramyxoviruses with a G protein do not bind sialic acid but instead bind to cellular factors such as 62 on Jauary 9, 2018 by gest http/jvi.asm .rg/ D ow nladed fom Ephrin B2 for the henipaviruses (8, 42). Members of the pneumovirinae subfamily express a functionally 63 different G protein which has been shown to interact with cell surface proteoglycans in the case of RSV 64 and HMPV (32, 66). Although it has been shown that most paramyxoviruses require the attachment 65 protein for binding and infection, a role for HMPV G protein in receptor binding has not been confirmed. 66 Interestingly, while the attachment protein is essential for virus attachment and subsequent membrane 67 fusion in the paramyxovirinae subfamily, studies have shown that some members of the pneumovirinae 68 subfamily can be infectious in the absence of the attachment protein. RSV lacking G can be propagated 69 in vitro but could not replicate efficiently in vivo (22, 64) and bovine respiratory syncytial virus (BRSV) 70 lacking G can still infect its host (52). Similarly, a recombinant avian metapneumovirus (AMPV), the 71 closest relative of HMPV, lacking the SH and G proteins was able to grow, albeit slower than wild-type 72 AMPV, in cell culture (41). Studies from our laboratory and others indicate that the G protein of HMPV is 73 also dispensable for attachment and fusion, as cell-cell fusion can be accomplished in the absence of G 74 and recombinant HMPV particles lacking G are infectious in vitro (54). Furthermore, a mutant virus 75 devoid of the G protein can efficiently infect African green monkeys (6) suggesting that the F protein of 76 HMPV is capable of performing both the attachment and fusion steps in vivo. Indeed, the HMPV F 77 protein has been shown to be an important marker of human infection (48). 78 Most cells express a large number of different surface carbohydrates that mediate multiple functions 79 such as adhesion, growth and signaling (50). The glycan synthesis of glycoproteins is based on two main 80 processes: N-glycosylation, which involves an addition of N-acetylglucosamine (GlcNAc) to the nitrogen 81 atom of an aspargine residue, or O-glycosylation, which is the addition of N-acetylgalactosamine 82 (GalNAc) or xylose to the oxygen atom of a serine or a threonine residue. GalNAc addition leads to the 83 synthesis of mucins, whereas xylose addition leads to synthesis of glycosaminoglycans (GAG). GAGs can 84 on Jauary 9, 2018 by gest http/jvi.asm .rg/ D ow nladed fom be classified into six different groups based on their composition: chondroitin sulfate, heparan sulfate, 85 dermatan sulfate, keratan sulfate, hyaluran, and heparin (3, 49, 61). 86 Heparan sulfate (HS) is the most often implicated GAG in virus-cell interactions. HS has been described 87 as a receptor for multiple herpesviruses and is also necessary during the first step of adhesion of other 88 viruses such as HIV and influenza (reviewed in (57)). HS plays an important role in the initial steps of RSV 89 entry, the closest related human pathogen to HMPV (9, 25, 26, 32). This interaction, however, appears 90 to be largely mediated by RSV G, and viruses lacking G show a significant decrease in binding to CHO-K1 91 cells (64). A recent study showed that the HMPV G protein also has the ability to bind GAGs and 92 suggested that this interaction might be a significant factor for virus-cell interactions (66). 93 Integrins are extracellular matrix binding proteins expressed in nearly all cell types and play multiple 94 roles including the regulation of cell adhesion, tissue growth, and migration (reviewed in (2)). These 95 heterodimers, composed of at least 18 different α and 10 different β subunits, bind to a myriad of 96 proteins including HS proteoglycans (HSPG), and many proteins that express the amino acid sequence 97 Arg-Gly-Asp (RGD) (51). Due to their broad distribution on the surfaces of many cell types, several 98 integrins have been implicated in viral entry. Integrins are part of the receptor complex of some 99 picornaviruses, hantaviruses, and papillomaviruses, and are also involved in adeno-associated virus, 100 Ebola virus, and reovirus entry, though not as cellular receptors (17, 39, 53, 60). Furthermore, it has 101 recently been reported that the αvβ1 integrin plays a major role in promoting HMPV infection (16). 102 In this study, we investigated the role of GAGs and the integrin αvβ1 in HMPV infection using wild-type 103 HMPV, a mutant virus lacking the G protein, and a mutant virus devoid of G and SH (6). We show that 104 the αvβ1 integrin is not essential for HMPV F-mediated fusion or for the initial binding step. Rather, our 105 data indicate that integrins promote infection at a step downstream of the initial binding. Additionally, 106 we demonstrate that the G and SH proteins do not play a major role in viral attachment, making F the 107 on Jauary 9, 2018 by gest http/jvi.asm .rg/ D ow nladed fom main viral determinant for HMPV binding. Finally, we show that HMPV F interacts specifically with 108 heparan sulfate and that HS is an indispensable component of the HMPV binding receptor complex. 109