Atypical Fusion Peptide of Nelson Bay Virus Fusion-Associated Small Transmembrane Protein

ABSTRACT A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.

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Genetic Analysis of Determinants for Spike Glycoprotein Assembly into Murine Coronavirus Virions: Distinct Roles for Charge-Rich and Cysteine-Rich Regions of the Endodomain

Journal of Virology ◽

10.1128/jvi.78.18.9904-9917.2004 ◽

2004 ◽

Vol 78 (18) ◽

pp. 9904-9917 ◽

Cited By ~ 42

Author(s):

Rong Ye ◽

Cynthia Montalto-Morrison ◽

Paul S. Masters

Keyword(s):

Transmembrane Domain ◽

Mutational Analysis ◽

Hepatitis Virus ◽

Transmembrane Protein ◽

Terminal Segment ◽

Type I ◽

Viral Fusion ◽

S Protein ◽

Infected Cells ◽

Carboxy Terminal

ABSTRACT The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.

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Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

Journal of Virology ◽

10.1128/jvi.77.18.9769-9779.2003 ◽

2003 ◽

Vol 77 (18) ◽

pp. 9769-9779 ◽

Cited By ~ 35

Author(s):

Maya Shmulevitz ◽

Jayme Salsman ◽

Roy Duncan

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Transmembrane Domain ◽

Nonstructural Protein ◽

Fusion Reaction ◽

Specific Interaction ◽

Syncytium Formation ◽

Surface Expression ◽

Fusion Activity ◽

Basic Region

ABSTRACT Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo- or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.

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Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

Journal of Virology ◽

10.1128/jvi.00573-07 ◽

2007 ◽

Vol 81 (18) ◽

pp. 10151-10160 ◽

Cited By ~ 59

Author(s):

Xiaohong Shi ◽

Alain Kohl ◽

Ping Li ◽

Richard M. Elliott

Keyword(s):

Reverse Genetics ◽

Virus Assembly ◽

Nonstructural Protein ◽

Cytoplasmic Tail ◽

Syncytium Formation ◽

Surface Expression ◽

Cell Surface Expression ◽

Type I ◽

Glycoprotein Precursor

ABSTRACT The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.

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Two Domains of the Erythropoietin Receptor Are Sufficient for Jak2 Binding/Activation and Function

Molecular and Cellular Biology ◽

10.1128/mcb.01035-06 ◽

2006 ◽

Vol 26 (22) ◽

pp. 8527-8538 ◽

Cited By ~ 34

Author(s):

Stéphane Pelletier ◽

Sébastien Gingras ◽

Megumi Funakoshi-Tago ◽

Sherié Howell ◽

James N. Ihle

Keyword(s):

Signal Transduction ◽

Biological Activity ◽

Transmembrane Domain ◽

Mutational Analysis ◽

Surface Expression ◽

Proximal Region ◽

Erythropoietin Receptor ◽

Cell Surface Expression ◽

And Function ◽

Mutant Receptors

ABSTRACT Biochemical and genetic studies have shown that Jak2 is an essential component of EpoR signal transduction which is required for normal erythropoiesis. However, whether Jak2 is the sole direct mediator of EpoR signal transduction remains controversial. To address this issue, we have used an extensive and systematic mutational analysis across the EpoR cytoplasmic tail and transmembrane domain with the goal of determining whether mutants that negatively affected EpoR biological activity but retained Jak2 activation could be identified. Analysis of over 40 mutant receptors established that two large domains in the membrane-proximal region, which include the previously defined Box1 and Box2 domains as well as a highly conserved glycine among cytokine receptors, are required for Jak2 binding and activation and to sustain biological activity of the receptor. Importantly, none of the mutants that lost the ability to activate Jak2 retained the ability to bind Jak2, thus questioning the validity of models of receptor reorientation for Jak2 activation. Also, no correlation was made between cell surface expression of the receptor and its ability to bind Jak2, thus questioning the role of Jak2 in trafficking the receptor to the plasma membrane. Collectively, the results suggest that Jak2 is the sole direct signaling molecule downstream of EpoR required for biological activity.

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Co-translational folding of the first transmembrane domain of ABC-transporter CFTR is supported by assembly with the first cytosolic domain

10.1101/2020.12.19.423590 ◽

2020 ◽

Author(s):

Bertrand Kleizen ◽

Marcel van Willigen ◽

Marjolein Mijnders ◽

Florence Peters ◽

Magda Grudniewska ◽

...

Keyword(s):

Transmembrane Domain ◽

Activity Cycle ◽

Surface Expression ◽

Cell Surface Expression ◽

Type I ◽

Inherited Disease ◽

Domain Specific ◽

Independent Events ◽

Cytosolic Domains ◽

And Function

ABSTRACTABC-transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography, with regulation driven by the cytosolic domains, and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the protein defective in the most abundant rare inherited disease cystic fibrosis, by combining biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies a different mode of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.

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Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity

Journal of Virology ◽

10.1128/jvi.72.11.8747-8755.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8747-8755 ◽

Cited By ~ 17

Author(s):

Decheng Yang ◽

Dorothy Hwang ◽

Zhiyong Qiu ◽

Shirley Gillam

Keyword(s):

Cell Surface ◽

Membrane Fusion ◽

Rubella Virus ◽

Surface Expression ◽

Cell Surface Expression ◽

Fusion Activity ◽

Wild Type ◽

Hydrophobic Region ◽

Hydrophobic Domain ◽

Infected Cells

ABSTRACT Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.

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Canine Influenza Virus is Mildly Restricted by Canine Tetherin Protein

Viruses ◽

10.3390/v10100565 ◽

2018 ◽

Vol 10 (10) ◽

pp. 565

Author(s):

Yun Zheng ◽

Xiangqi Hao ◽

Qingxu Zheng ◽

Xi Lin ◽

Xin Zhang ◽

...

Keyword(s):

Influenza Virus ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Cellular Damage ◽

Interferon Response ◽

Type I ◽

Canine Influenza Virus ◽

Immune Factor ◽

Canine Influenza ◽

The Impact

Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell ·defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-α. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

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Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

Journal of Molecular Endocrinology ◽

10.1530/jme-12-0161 ◽

2012 ◽

Vol 49 (3) ◽

pp. 237-248 ◽

Cited By ~ 21

Author(s):

Hui Huang ◽

Ya-Xiong Tao

Keyword(s):

Cell Surface ◽

Structure Function ◽

Transmembrane Domain ◽

Mapk Pathway ◽

Surface Expression ◽

Camp Signaling ◽

Cell Surface Expression ◽

Melanocortin 4 Receptor ◽

Function Relationship ◽

Relationship Of

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

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