Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294

Yun, Bingling; Guan, Xi‐Wen; Liu, Yongzhen; Gao, Yanni; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Li; Li, Kai; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

doi:10.1038/srep15584

Cited by 14 publications

(24 citation statements)

References 58 publications

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“…In a previous study, we demonstrated that the cleavage of aMPV/B F protein is independent of exogenous trypsin (34). Here, we investigated the effect of adding trypsin on replication of aMPV/B.…”

Section: Ampv/b Grows In Vitro Without the Addition Of Trypsinmentioning

confidence: 93%

“…Syncytium formation assay to measure fusogenicity of the F protein. The syncytium formation assay was performed to analyze F protein fusogenic activity as previously described (34)(35)(36). Vero cells grown in 6-well plates to 85% confluence were transfected with 1 g plasmid carrying the F gene with ExFect transfection reagent (Vazyme Biotech, Nanjing, China) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

“…The areas forming syncytia were analyzed with a Magnetic Lasso tool and the histogram function of Adobe Photoshop software. The ratio of the area forming syncytia to the total area was calculated to estimate the fusogenicity of F protein as previously described (34)(35)(36). To study the effect of TMPRSS12 on fusogenic activity of F protein, TMPRSS12 was selectively overexpressed or knocked down in Vero cells before transfection with the plasmid carrying the F gene.…”

Section: Methodsmentioning

confidence: 99%

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TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus

Yun

Zhang

Liu

et al. 2016

J Virol

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The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. A vian metapneumovirus (aMPV), a member of the genus Metapneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae, causes acute rhinotracheitis (TRT) in turkeys and swollen head syndrome (SHS) in chickens (1-3). Thus, aMPV is a major disease threat to the turkey and chicken industries (4, 5). However, no effective vaccine or drug is currently available for large commercial poultry producers to prevent or treat aMPV (6). Based on a phylogenetic analysis, aMPV is divided into four subtypes: aMPV/A, aMPV/B, aMPV/C, and aMPV/D (7, 8). IMPORTANCE Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV in-The entry of paramyxoviruses into host cells initially requires the fusion of viral and cell membranes, which is mediated by one or more viral glycoproteins on the exterior of the viral envelope (9-12). For metapneumoviruses like aMPV, fusion (F) protein alone, without the help of an attachment protein, can induce membrane fusion (13-16). Consistent with this observation, recombinant aMPV lacking the small hydrophobic (SH) and attachment (G) glycoproteins replicates efficiently in vivo and vitro (17,18). Collectively, these results suggest that aMPV F protein alone can mediate membrane fusion and viral infection.The prerequisite for paramyxovirus F protein mediation of membrane fusion is cleavage of the F protein, followed by a conformational change (19)(20)(21)(22). The paramyxovirus F protein is synthesized as a full-length precursor (F0) that is subsequently cleaved by cellular proteases to generate F1 and F2. Cleavage of F0 exposes a fusion peptide at the N terminus of F1 that is responsible for mediating membrane fusi...

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Section: Ampv/b Grows In Vitro Without the Addition Of Trypsinmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus

Yun

Zhang

Liu

et al. 2016

J Virol

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“…The entry of paramyxoviruses into the cell is thought to occur at the cell surface in a low pH-independent manner, a mechanism that requires the binding of the envelope glycoproteins to its receptors to activate membrane fusion of the virus and cell (38). Our prior work has demonstrated that residue His 294 is responsible for aMPV/B F protein-mediated cell-cell fusion independent of low pH (42), suggesting that the aMPV/B F protein attachment to integrin leads to cell-surface fusion. Further investigation is needed to determine whether the entry of aMPV/B occurs at the cell surface.…”

Section: Discussionmentioning

confidence: 99%

“…We previously demonstrated that the aMPV F protein alone can induce cell-cell fusion (41,42), suggesting that aMPV F protein has the function for binding to its receptor(s). A series of sequence/structure analysis, blockade, knockdown, overexpression, and mutagenesis experiments demonstrated that integrin is a functional receptor for the aMPV/B F protein, which induces cell-cell fusion and aMPV/B infection.…”

Section: Discussionmentioning

confidence: 99%

Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection

Yun

Guan

Liu

et al. 2016

Journal of Biological Chemistry

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Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin ␣v and/or ␤1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin ␣v and ␤1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin ␣v␤1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. Avian metapneumovirus (aMPV)3 belongs to the genus Metapneumovirus in the subfamily Pneumovirinae of paramyxoviridae (1, 2). aMPV is a major cause of acute rhinotracheitis in turkeys and is associated with swollen head syndrome in chickens. Thus, aMPV is considered a major threat to the poultry industry (1, 2). Based on phylogenetic analysis, aMPV has been divided into four subtypes: aMPV/A, aMPV/B, aMPV/C, and aMPV/D (3).The entry of a paramyxovirus into the host cell initially requires fusion of the viral envelope and cellular membrane, which is mediated by the glycoproteins on the viral envelope (4 -6). For viruses of the Paramyxovirinae subfamily, such as Newcastle disease virus, fusion (F) protein-mediated membrane fusion requires the assistance of the hemagglutininneuraminidase (HN) protein (7-10). However, viruses in the Pneumovirinae subfamily including human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and aMPV, the F protein alone in the absence of the small hydrophobic and attachment (G) proteins can mediate membrane fusion and viral infection (11-15), suggesting that the mechanism by which hRSV, hMPV, and aMPV mediate virus-cell membrane fusion might be unique among the Paramyxovirinae subfamily.The F protein of Paramyxovirinae viruses is activated by a non-F attachment glycoprotein bound to its receptor(s) on the host cell (10, 16). The F protein then undergoes a coordinated series of conformational changes to its most stable form to promote membrane fusion (17-20). Thus, binding of the attachment glycoprotein to the cellular receptor(s) is critical for triggering the fusion process (21,22). For example, Newcastle disease virus HN protein attaches to sialic acid-containing receptors on the host cell surface and then activates the F protein to induce membrane fu...

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