scholarly journals Pseudorevertants of a Semliki Forest Virus Fusion-Blocking Mutation Reveal a Critical Interchain Interaction in the Core Trimer

2010 ◽  
Vol 84 (22) ◽  
pp. 11624-11633 ◽  
Author(s):  
Catherine Y. Liu ◽  
Christen Besanceney ◽  
Yifan Song ◽  
Margaret Kielian
Keyword(s):  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Critical Role ◽  
Salt Bridges ◽  
Growth Defects ◽  
The Core ◽  
Adjacent Chain ◽  
Virus Fusion ◽  
Important Interaction ◽  
Domain Iii

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-like homotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein.

scholarly journals Functions of the Stem Region of the Semliki Forest Virus Fusion Protein during Virus Fusion and Assembly

2006 ◽  
Vol 80 (22) ◽  
pp. 11362-11369 ◽  
Author(s):  
Maofu Liao ◽  
Margaret Kielian
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Protein A ◽  
Stem Length ◽  
Membrane Fusion Protein ◽  
Virus Fusion ◽  
Target Membrane ◽  
Domain Iii

ABSTRACT Membrane fusion of the alphaviruses is mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 dissociates from its heterodimer interaction with the E2 protein and forms a target membrane-inserted E1 homotrimer. The structure of the homotrimer is that of a trimeric hairpin in which E1 domain III and the stem region fold back toward the target membrane-inserted fusion peptide loop. The E1 stem region has a strictly conserved length and several highly conserved residues, suggesting the possibility of specific stem interactions along the trimer core and an important role in driving membrane fusion. Mutagenesis studies of the alphavirus Semliki Forest virus (SFV) here demonstrated that there was a strong requirement for the E1 stem in virus assembly and budding, probably reflecting its importance in lateral interactions of the envelope proteins. Surprisingly, however, neither the conserved length nor any specific residues of the stem were required for membrane fusion. Although the highest fusion activity was observed with wild-type E1, efficient fusion was mediated by stem mutants containing a variety of substitutions or deletions. A minimal stem length was required but could be conferred by a series of alanine residues. The lack of a specific stem sequence requirement during SFV fusion suggests that the interaction of domain III with the trimer core can provide sufficient driving force to mediate membrane merger.

scholarly journals A Conserved Histidine in the ij Loop of the Semliki Forest Virus E1 Protein Plays an Important Role in Membrane Fusion

2004 ◽  
Vol 78 (24) ◽  
pp. 13543-13552 ◽  
Author(s):  
Chantal Chanel-Vos ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Infectious Clone ◽  
Critical Role ◽  
Low Ph ◽  
Sequence Comparisons ◽  
E1 Protein ◽  
Fusion Loop

ABSTRACT The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered membrane fusion reaction mediated by the E1 protein. E1 is a class II fusion protein that contains the hydrophobic fusion peptide loop and converts to a stable homotrimer during the fusion reaction. Intriguingly, the fusion loop is closely associated with a loop connecting the i and j β-strands. This ij loop plays a role in the cholesterol dependence of membrane fusion and is specifically susceptible to proteolysis in the protease-resistant E1 homotrimer. The SFV ij loop contains a histidine residue at position 230. Sequence comparisons revealed that an analogous histidine is completely conserved in all alphavirus and flavivirus fusion proteins. An E1 H230A mutant was constructed using the SFV infectious clone. Although cells infected with H230A RNA produced virus particles, these virions were completely noninfectious and were blocked in both cell-cell fusion and lipid mixing assays. The H230A virions efficiently bound to cell surface receptors and responded to low pH by undergoing acid-dependent conformational changes including dissociation of the E1/E2 dimer, exposure of the fusion loop, association with target liposomes, exposure of acid-conformation-specific epitopes, and formation of the stable E1 homotrimer. Studies with a soluble fragment of E1 showed that the mutant protein was defective in lipid-dependent conformational changes. Our results indicate that the E1 ij loop and the conserved H230 residue play a critical role in alphavirus-membrane fusion and suggest the presence of a previously undescribed late intermediate in the fusion reaction.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Spike protein oligomerization control of Semliki Forest virus fusion.

1990 ◽  
Vol 64 (10) ◽  
pp. 5214-5218 ◽  
Author(s):  
M Lobigs ◽  
J M Wahlberg ◽  
H Garoff
Keyword(s):  
Semliki Forest Virus ◽  
Spike Protein ◽  
Protein Oligomerization ◽  
Virus Fusion

Evaluation on the performance fluctuation after water ingestion for a turbofan engine compressor during flight descent

2020 ◽  
pp. 095441002097798
Author(s):  
Lu Yang ◽  
Qun Zheng ◽  
Aqiang Lin
Keyword(s):  
Droplet Diameter ◽  
Pressure Ratio ◽  
Critical Role ◽  
Water Film ◽  
Heavy Rain ◽  
Diameter Distribution ◽  
Turbofan Engine ◽  
Water Ingestion ◽  
The Core ◽  
Engine Compressor

Turbofan engine compressor is most severely threatened by the entry of liquid water during flight descent. This study aims to deeply understand the fluctuations of compressor performance parameters caused by water ingestion through frequency spectrum analysis. The water content and droplet diameter distribution are determined based on the real heavy rain environment. Results reveal that most of the droplets actually entering the core compressor have a particle size of less than 100 μm. In addition, the formation and motion of water film plays a critical role in affecting the fluctuation characteristics. Water ingestion deteriorates the compression performance and aggravates the unsteady fluctuations of the fan. However, the performance of the core compressor is less affected by water ingestion, but their fluctuations are still exacerbated. For some important parameters, such as inlet mass flow rate, total pressure ratio, total temperature ratio, compression work and efficiency, their main frequency of fluctuation are switched from the original blade passing frequency to the rotor passing frequency, and their amplitudes are correspondingly amplified to varying degrees. These phenomena can be observed in both the fluctuations of the fan and core compressor. Moreover, the operating point of them will be in the long-period and large-amplitude fluctuations, which leads them experiences the non-optimal state for a long time and threatens their operating stability.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

scholarly journals A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane.

1986 ◽  
Vol 103 (5) ◽  
pp. 1829-1835 ◽  
Author(s):  
P G Woodman ◽  
J M Edwardson
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Trichloroacetic Acid ◽  
Plasma Membranes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Viral Glycoprotein ◽  
Acetylneuraminic Acid ◽  
Donor And Acceptor ◽  
A Cell

A cell-free assay has been developed for the delivery of influenza virus neuraminidase to the plasma membrane. Two types of postnuclear supernatant, which acted as donor and acceptor of the enzyme, were prepared from baby hamster kidney cells. Donor preparations were obtained from cells infected with influenza virus and containing neuraminidase en route to the plasma membrane. Acceptor preparations were obtained from cells containing, bound to their plasma membranes, Semliki Forest virus with envelope glycoproteins bearing [3H]N-acetylneuraminic acid. Fusion between vesicles from these two preparations permits access of the enzyme to its substrate, which results in the release of free [3H]N-acetylneuraminic acid. This release was detected through the transfer of radioactivity from a trichloroacetic acid-insoluble to a trichloroacetic acid-soluble fraction. An ATP-dependent component of release was found, which appears to be a consequence of vesicle fusion. This component was enhanced when the donor was prepared from cells in which the enzyme had been concentrated in a compartment between the Golgi complex and the plasma membrane, which indicates that a specific exocytic fusion event has been reconstituted. The extent of fusion is greatly reduced by pre-treatment of donor and acceptor preparations with trypsin, which points to the involvement of proteins in the fusion reaction.

Revising the Assumption that Ḥadīṯ Studies Flourished in the 11th/17th-Century Ḥiǧāz: Ibrāhīm al-Kūrānī’s (d. 1101/1690) Contribution

Arabica ◽  
2021 ◽  
Vol 68 (1) ◽  
pp. 1-35
Author(s):  
Naser Dumairieh
Keyword(s):  
General Framework ◽  
18Th Century ◽  
Critical Role ◽  
17Th Century ◽  
Main Interest ◽  
Islamic World ◽  
The Core ◽  
Revival Movement ◽  
Almost All

Abstract The Ḥiǧāz in the 11th/17th century has long been considered the center of a “revival” movement in ḥadīṯ studies. This assumption has spread widely among scholars of the 11th-/17th- and 12th-/18th-century Islamic world based on the fact that the isnāds of many major ḥadīṯ scholars from almost all parts of the Islamic world from the 11th/17th century onward return to a group of scholars in the Ḥiǧāz. The scholarly group that is assumed to have played a critical role in the flourishing of ḥadīṯ studies in the 11th/17th-century Ḥiǧāz is called the al-Ḥaramayn circle or network. However, to date, there have been no studies that investigate what was actually happening in that century concerning ḥadīṯ studies. Examining the actual ḥadīṯ studies of one of the scholars at the core of al-Ḥaramayn circle, i.e. Ibrāhīm b. Ḥasan al-Kūrānī, will unpack the main interest of Ḥiǧāzī scholars in ḥadīṯ literature, reveal previously unstudied aspects of ḥadīṯ studies in the 11th/17th-century Ḥiǧāz, correct some unexamined assumptions, and situate the ḥadīṯ efforts of scholars of the 11th/17th-century Ḥiǧāz within a general framework of developments within ḥadīṯ studies.

The Dialectics of Resistance: Colonial Geography, Bengali Literati and the Racial Mapping of Indian Identity

2009 ◽  
Vol 44 (1) ◽  
pp. 53-79 ◽  
Author(s):  
SUBHO BASU
Keyword(s):  
Nineteenth Century ◽  
Information System ◽  
Geographic Information System ◽  
Critical Role ◽  
Geographic Information ◽  
Indian Society ◽  
Nation State ◽  
Colonial India ◽  
The Core ◽  
Indian Identity

AbstractThrough a study of hitherto unexplored geography textbooks written in Bengali between 1845 and 1880, this paper traces the evolution of a geographic information system related to ethnicity, race, and space. This geographic information system impacted the mentality of emerging educated elites in colonial India who studied in the newly established colonial schools and played a critical role in developing and articulating ideas of the territorial nation-state and the rights of citizenship in India. The Bengali Hindu literati believed that the higher location of India in such a constructed hierarchy of civilizations could strengthen their claims to rights of citizenship and self-government. These nineteenth century geography textbooks asserted clearly that high caste Hindus constituted the core ethnicity of colonial Indian society and all others were resident outsiders. This knowledge system, rooted in geography/ethnicity/race/space, and related to the hierarchy of civilizations, informed the Bengali intelligentsia's notion of core ethnicity in the future nation-state in India with Hindu elites at its ethnic core.

scholarly journals Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes

1999 ◽  
Vol 73 (10) ◽  
pp. 8476-8484 ◽  
Author(s):  
Jolanda M. Smit ◽  
Robert Bittman ◽  
Jan Wilschut
Keyword(s):  
Sindbis Virus ◽  
Core Protein ◽  
Fusion Reaction ◽  
Low Ph ◽  
Viral Envelope ◽  
Cell Entry ◽  
Receptor Interaction ◽  
Neutral Ph ◽  
Virus Fusion ◽  
Entry Mechanism

ABSTRACT There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.

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