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Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

ABSTRACT: Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins, the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins, and as extension of previous work (Palomo et al., 2014), a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion.

SUBMITTER: Rodriguez L

PROVIDER: S-EPMC7119586 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

Rodríguez Laura L Olmedillas Eduardo E Mas Vicente V Vázquez Mónica M Cano Olga O Terrón María C MC Luque Daniel D Palomo Concepción C Melero José A JA

Journal of virological methods 20150812

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins, the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle ...[more]

PMID: 26275682

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Project description:Langya virus (LayV) is a paramyxovirus in the Henipavirus genus, closely related to the deadly Nipah (NiV) and Hendra (HeV) viruses, that was identified in August 2022 through disease surveillance following animal exposure in eastern China. Paramyxoviruses present two glycoproteins on their surface, known as attachment and fusion proteins, that mediate entry into cells and constitute the primary antigenic targets for immune response. Here, we determine cryo-electron microscopy (cryo-EM) structures of the uncleaved LayV fusion protein (F) ectodomain in pre- and postfusion conformations. The LayV-F protein exhibits pre- and postfusion architectures that, despite being highly conserved across paramyxoviruses, show differences in their surface properties, in particular at the apex of the prefusion trimer, that may contribute to antigenic variability. While dramatic conformational changes were visualized between the pre- and postfusion forms of the LayV-F protein, several domains remained invariant, held together by highly conserved disulfides. The LayV-F fusion peptide (FP) is buried within a highly conserved, hydrophobic interprotomer pocket in the prefusion state and is notably less flexible than the rest of the protein, highlighting its "spring-loaded" state and suggesting that the mechanism of pre-to-post transition must involve perturbations to the pocket and release of the fusion peptide. Together, these results offer a structural basis for how the Langya virus fusion protein compares to its Henipavirus relatives and propose a mechanism for the initial step of pre- to postfusion conversion that may apply more broadly to paramyxoviruses. IMPORTANCE The Henipavirus genus is quickly expanding into new animal hosts and geographic locations. This study compares the structure and antigenicity of the Langya virus fusion protein to other henipaviruses, which have important vaccine and therapeutic development implications. Furthermore, the study proposes a new mechanism to explain the early steps of the fusion initiation process that can be more broadly applied to the Paramyxoviridae family.

Project description:Retroviral elements from endogenous retroviruses have functions in mammalian physiology. The best-known examples are the envelope proteins that function in placenta development and immune suppression. Porcine endogenous retroviruses (PERVs) are an understudied class of endogenous retroviruses that infect cultured human cells, raising concern regarding porcine xenografts. The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development. Despite the growing interest in PERVs, their envelope glycoproteins remain poorly characterized. Here, we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain. The PERV fusion protein structure reveals a conserved class I viral fusion protein six-helix bundle. Biophysical experiments demonstrated that the thermodynamic stability of the PERV fusion protein secondary structure was the same at physiological and acidic pHs. A conserved surface analysis highlights the high degree of sequence conservation among retroviral fusogens in the chain reversal region that facilitates the large-scale conformational change required for membrane fusion. Further structural alignment of class I viral fusogens revealed a phylogenetic clustering that shows evolution into various lineages that correlate with virus mechanisms of cell entry. Our work indicates that structural dendrograms can be used to qualitatively infer insights into the fusion mechanisms of newly discovered class I viral fusogen structures. IMPORTANCE Class I viral fusion proteins represent a diverse group of fusogens that catalyze membrane fusion. Although structural studies have focused on those from exogenous viruses, ancient retroviral infections of germ line cells have immortalized ancient fusogens in eukaryotic genomes. These "fossilized" glycoproteins are poorly defined compared to modern fusogens. In this study, we characterized and determined the structure of the porcine endogenous retrovirus fusogen, an ancient retroviral element captured by swine. This fusion protein revealed remarkable alignment to exogenous retroviral fusion proteins, suggesting that fossil fusogens utilize similar structural determinants to perform membrane fusion. Moreover, structural phylogenetic analysis demonstrates that class I viral fusogens cluster into distinct lineages defined by mechanism of membrane fusion. Our results suggest that structural dendrograms can be used to infer mechanistic insights for uncharacterized fusion proteins.

Dataset Information

Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

Publications

Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

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