The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

Abstract

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...

Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

“…1B). The SH-GFP fusion protein was observed in multiple molecular weight bands, consistent with glycosylated and unglycosylated forms of SH as described previously (Masante et al, 2014), suggesting physiologic protein expression and processing (Supplementary Fig. 1B).…”

“…Sequence homology between HMPV SH and related viruses is very low (Biacchesi et al, 2004a; Yunus et al, 2003) but maintains key characteristics such as similar hydrophilicity and a high percentage of threonine and serine residues (Piyaratna et al, 2011). Recent data indicate that, similar to the RSV SH protein, the HMPV SH protein is capable of forming a viroporin and increasing cell permeability (Masante et al, 2014). This is particularly intriguing in the context of our work, because dephosphorylated STAT1 has been shown to interact with the host nucleoporins, Nup153 and Nup214, in order to translocate out of the nucleus after signaling (Marg et al, 2004).…”

Partial Attenuation of Respiratory Syncytial Virus with a Deletion of a Small Hydrophobic Gene Is Associated with Elevated Interleukin-1β Responses

“…SH genes are found in all members of the subfamily Pneumovirinae and some of the Paramyxovirinae, including mumps virus (48) and human parainfluenza virus 5 (hPIV5) (49). The SH proteins from human metapneumovirus (hMPV) (50), bRSV (51), hPIV5 (49), and mumps virus (52) are nonessential for in vitro growth, and an immunomodulatory function has been described for the SH proteins from PIV5 (49), bRSV (17), and hMPV (53), which also encodes a viroporin (54). We hypothesize that the pore structure of the SH protein (10) facilitates this immunomodulatory function, counteracting the activation of NLRP3 by ionic changes induced by RSV infection (27).…”

Emerging Roles of Inflammasomes in Acute Pneumonia

“…The small hydrophobic protein of the metapneumovirus was found to act similarly to a viroporin and can affect membrane permeability (60). In another study in children, Il-1b, Il-18, Nlrp3, and IkBa genes were upregulated in metapneumovirus infection.…”