Insect High Five cells (Invitrogen) were maintained in Express 5 serum-free medium (Gibco) supplemented with 10% (vol/vol) GlutaMax (Gibco) and 1% penicillin/streptomycin

Insect High Five cells (Invitrogen) were maintained in Express 5 serum-free medium (Gibco) supplemented with 10% (vol/vol) GlutaMax (Gibco) and 1% penicillin/streptomycin. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced Ro 61-8048 triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event. The family of enveloped viruses includes many medically and economically important pathogens (1). This large family of negative-sense, single-stranded RNA viruses includes measles virus (MeV), mumps virus, human parainfluenza viruses 1C4 (hPIV1C4), parainfluenza virus 5 (PIV5, formerly known as simian virus 5, SV5), human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), Newcastle disease virus (NDV), Sendai virus, and canine distemper virus (CDV). Newly discovered and highly pathogenic paramyxoviruses Ro 61-8048 include the zoonotic Nipah and Hendra viruses. The recent discovery of 66 unique mammalian paramyxovirus Rabbit Polyclonal to SLC25A12 sequences from bats and rodents (2) suggests that the impact of this virus family on human health will continue to grow. Paramyxoviruses, like all enveloped viruses, possess a lipid bilayer that is derived from the host cell during viral egress. Paramyxovirus particles must fuse their membrane with the membrane of a target host cell for successful infection to occur. Embedded in the viral envelope are the viral spike glycoproteins necessary for receptor binding and fusion with a target cell. The receptor-binding and fusion activities are carried out by a variable attachment protein, known as HN, H, or G protein, and a more conserved fusion (F) protein, respectively (3C5). Coexpression of HN, H, or G and the F protein in the same cell is required to activate the F protein, and coimmunoprecipitation data indicate F and HN, H, or G interact physically through the HN, H, or G stalk region (5C13). Receptor specificity of the attachment protein (14C19) ensures that the F protein is activated Ro 61-8048 at the correct time and place for a successful invasion of a target cell. Although F protein activation is mediated naturally via interaction with the HN, H, or G stalk domain in most paramyxovirus species (20C27), increased temperature can be used as a surrogate trigger for PIV5, CDV, and MeV F proteins (24, 28C32). F proteins from different paramyxoviruses have different energy requirements for activation (24, 29, 33, 34). Paramyxovirus F proteins, together with influenza virus HA, Ebola virus GP, and HIV Env, among others, are classified as class I viral fusion proteins (35). Class I fusion proteins share the following characteristics: all are trimers, all initially fold in the endoplasmic reticulum into a metastable prefusion conformation, and all are synthesized as a precursor that must be cleaved to generate a new N terminus that is hydrophobic and known as the fusion peptide (FP). Cleavage usually is required for membrane fusion, viral infectivity, and pathogenicity (36C39). The current model of class I fusion protein function posits that the metastable F protein, upon activation by HN, H, Ro 61-8048 or G, undergoes a series of large-scale refolding events, going down an energy gradient from a metastable prefusion form to a final, stable postfusion form (reviewed in refs. 3, 5, and 40). The work done in refolding brings the lipid bilayers in close proximity so that membrane merger (fusion) can occur. Atomic structures have been obtained for PIV5 and hRSV F proteins in the Ro 61-8048 prefusion form (41C43) and for hPIV3, NDV, and hRSV F proteins in the postfusion form (44C47). Together with biochemical data and EM images (11, 48, 49), these.