scholarly journals Glycoprotein 3 of porcine reproductive and respiratory syndrome virus exhibits an unusual hairpin-like membrane topology

2018 ◽  
Author(s):  
Minze Zhang ◽  
Ludwig Krabben ◽  
Fangkun Wang ◽  
Michael Veit
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Membrane Topology ◽  
Respiratory Syndrome Virus ◽  
Amphipathic Helix ◽  
Hydrophobic Region ◽  
Virus Particles ◽  
Terminal Domain ◽  
C Terminus ◽  
Amino Acid Variability

ABSTRACTThe glycoprotein GP3 of the Arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported.We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells; GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C-terminus of GP3, fused to GFP, is resistant against proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C-terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3 and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region might form an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents and in its hydrophobic face enhances secretion of GP3. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.IMPORTANCEThe porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here we analyzed basic structural features of glycoprotein 3 (GP3) from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 might play a role during PRRSV infection of pigs; it might serve as a decoy to distract antibodies away from virus particles.

scholarly journals Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Minze Zhang ◽  
Ludwig Krabben ◽  
Fangkun Wang ◽  
Michael Veit
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Membrane Topology ◽  
Respiratory Syndrome Virus ◽  
Amphipathic Helix ◽  
Hydrophobic Region ◽  
Virus Particles ◽  
Terminal Domain ◽  
C Terminus ◽  
Amino Acid Variability

ABSTRACTGlycoprotein 3 (GP3) of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region, and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported. We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells, GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C terminus of GP3, fused to green fluorescent protein (GFP), is resistant to proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3, and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region forms an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents secretion of GP3 and in its hydrophobic face enhances it. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.IMPORTANCEPRRSV is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here, we analyzed basic structural features of GP3 from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 plays a role during PRRSV infection of pigs: it might serve as a decoy to distract antibodies away from virus particles.

scholarly journals Mutational Analysis of the Putative Fusion Domain of Ebola Virus Glycoprotein

1999 ◽  
Vol 73 (10) ◽  
pp. 8907-8912 ◽  
Author(s):  
Hiroshi Ito ◽  
Shinji Watanabe ◽  
Anthony Sanchez ◽  
Michael A. Whitt ◽  
Yoshihiro Kawaoka
Keyword(s):  
Amino Acids ◽  
Receptor Binding ◽  
Ebola Virus ◽  
Mutational Analysis ◽  
Structural Features ◽  
Fusion Peptide ◽  
Hydrophobic Region ◽  
Virus Particles ◽  
Membrane Fusion Protein ◽  
Vesicular Stomatitis

ABSTRACT Ebola viruses contain a single glycoprotein (GP) spike, which functions as a receptor binding and membrane fusion protein. It contains a highly conserved hydrophobic region (amino acids 524 to 539) located 24 amino acids downstream of the N terminus of the Ebola virus GP2 subunit. Comparison of this region with the structural features of the transmembrane subunit of avian retroviral GPs suggests that the conserved Ebola virus hydrophobic region may, in fact, serve as the fusion peptide. To test this hypothesis directly, we introduced conservative (alanine) and nonconservative (arginine) amino acid substitutions at eight positions in this region of the GP2 molecule. The effects of these mutations were deduced from the ability of the Ebola virus GP to complement the infectivity of a vesicular stomatitis virus (VSV) lacking the receptor-binding G protein. Some mutations, such as Ile-to-Arg substitutions at positions 532 (I532R), F535R, G536A, and P537R, almost completely abolished the ability of the GP to support VSV infectivity without affecting the transport of GP to the cell surface and its incorporation into virions or the production of virus particles. Other mutations, such as G528R, L529A, L529R, I532A, and F535A, reduced the infectivity of the VSV-Ebola virus pseudotypes by at least one-half. These findings, together with previous reports of liposome association with a peptide corresponding to positions 524 to 539 in the GP molecule, offer compelling support for a fusion peptide role for the conserved hydrophobic region in the Ebola virus GP.

scholarly journals Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

2001 ◽  
Vol 8 (3) ◽  
pp. 598-603 ◽  
Author(s):  
Sarah Wootton ◽  
Gabriella Koljesar ◽  
Liuzhan Yang ◽  
Kyoung-Jin Yoon ◽  
Dongwan Yoo
Keyword(s):  
Amino Acids ◽  
Secondary Structure ◽  
Nucleocapsid Protein ◽  
Conformational Epitope ◽  
Single Amino Acid ◽  
Respiratory Syndrome Virus ◽  
Beta Strand ◽  
N Protein ◽  
Terminal Domain ◽  
C Terminus

ABSTRACT Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.

scholarly journals The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

2003 ◽  
Vol 185 (13) ◽  
pp. 3821-3827 ◽  
Author(s):  
Elisabeth Enggist ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heme Binding ◽  
Membrane Anchor ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Helical Turn ◽  
Low Activity

ABSTRACT CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.

scholarly journals Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity

1995 ◽  
Vol 311 (2) ◽  
pp. 699-704 ◽  
Author(s):  
A Muraoka ◽  
M Hashiramoto ◽  
A E Clark ◽  
L C Edwards ◽  
H Sakura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Structural Features ◽  
Transport Activity ◽  
Wild Type ◽  
C Terminus ◽  
Wild Type Clone

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.

scholarly journals VP24 Is a Chitin-Binding Protein Involved in White Spot Syndrome Virus Infection

2015 ◽  
Vol 90 (2) ◽  
pp. 842-850 ◽  
Author(s):  
Zaipeng Li ◽  
Fang Li ◽  
Yali Han ◽  
Limei Xu ◽  
Feng Yang
Keyword(s):  
Amino Acids ◽  
Digestive Tract ◽  
Envelope Protein ◽  
White Spot Syndrome Virus ◽  
Viral Envelope ◽  
White Spot ◽  
Viral Envelope Protein ◽  
Virus Particles ◽  
C Terminus ◽  
Shrimp Farms

ABSTRACTOral ingestion is the major route of infection for the white spot syndrome virus (WSSV). However, the mechanism by which virus particles in the digestive tract invade host cells is unknown. In the present study, we demonstrate that WSSV virions can bind to chitin through one of the major envelope proteins (VP24). Mutagenesis analysis indicated that amino acids (aa) 186 to 200 in the C terminus of VP24 were required for chitin binding. Moreover, the P-VP24186–200peptide derived from the VP24 chitin binding region significantly inhibited the VP24-chitin interaction and the WSSV-chitin interaction, implying that VP24 participates in WSSV binding to chitin. Oral inoculation experiments showed that P-VP24186–200treatment reduced the number of virus particles remaining in the digestive tract during the early stage of infection and greatly hindered WSSV proliferation in shrimp. These data indicate that binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSVper osinfection and provide new ideas for preventing WSSV infection in shrimp farms.IMPORTANCEIn this study, we show that WSSV can bind to chitin through the envelope protein VP24. The chitin-binding domain of VP24 maps to amino acids 186 to 200 in the C terminus. Binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSVper osinfection. These findings not only extend our knowledge of WSSV infection but also provide new insights into strategies to prevent WSSV infection in shrimp farms.

scholarly journals Plasma Membrane and Nuclear Localization of G Protein–coupled Receptor Kinase 6A

2007 ◽  
Vol 18 (8) ◽  
pp. 2960-2969 ◽  
Author(s):  
Xiaoshan Jiang ◽  
Jeffrey L. Benovic ◽  
Philip B. Wedegaertner
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
G Protein ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Amphipathic Helix ◽  
G Protein Coupled Receptor ◽  
C Terminus ◽  
Acidic Residues ◽  
G Protein Coupled

G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

scholarly journals Stem-Loop Binding Protein Facilitates 3′-End Formation by Stabilizing U7 snRNP Binding to Histone Pre-mRNA

1999 ◽  
Vol 19 (5) ◽  
pp. 3561-3570 ◽  
Author(s):  
Zbigniew Dominski ◽  
Lian-Xing Zheng ◽  
Ricardo Sanchez ◽  
William F. Marzluff
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Binding Protein ◽  
Nuclear Extract ◽  
Stable Complex ◽  
Stem Loop ◽  
Terminal Domain ◽  
C Terminus ◽  
Loop Sequence ◽  
U7 Snrnp

ABSTRACT The 3′ end of histone mRNA is formed by an endonucleolytic cleavage of the primary transcript after a conserved stem-loop sequence. The cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3′-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine the regions of human SLBP necessary for 3′ processing, various deletion mutants of the protein were tested for their ability to complement the SLBP-depleted extract. The entire N-terminal domain and the majority of the C-terminal domain of human SLBP are dispensable for processing. The minimal protein that efficiently supports cleavage of histone pre-mRNA consists of 93 amino acids containing the 73-amino-acid RNA-binding domain and 20 amino acids located immediately next to its C terminus. Replacement of these 20 residues with an unrelated sequence in the context of the full-length SLBP reduces processing >90%. Coimmunoprecipitation experiments with the anti-SLBP antibody demonstrated that SLBP and U7 snRNP form a stable complex only in the presence of pre-mRNA substrates containing a properly positioned U7 snRNP binding site. One role of SLBP is to stabilize the interaction of the histone pre-mRNA with U7 snRNP.

scholarly journals Topogenesis in membranes of the NTB–VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain

2004 ◽  
Vol 85 (2) ◽  
pp. 535-545 ◽  
Author(s):  
Aiming Wang ◽  
Sumin Han ◽  
Hélène Sanfaçon
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Microsomal Membranes ◽  
Hydrophobic Amino Acids ◽  
Definition Of

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex.

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