scholarly journals A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

1998 ◽  
Vol 140 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Malini Vashishtha ◽  
Thomas Phalen ◽  
Marianne T. Marquardt ◽  
Jae S. Ryu ◽  
Alice C. Ng ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Single Point ◽  
Cellular Membrane ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Protein Subunit ◽  
Single Amino Acid Change

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

scholarly journals The Cholesterol Requirement for Sindbis Virus Entry and Exit and Characterization of a Spike Protein Region Involved in Cholesterol Dependence

1999 ◽  
Vol 73 (5) ◽  
pp. 4272-4278 ◽  
Author(s):  
Yanping E. Lu ◽  
Todd Cassese ◽  
Margaret Kielian
Keyword(s):  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Virus Entry ◽  
Single Point ◽  
Endocytic Pathway ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Entry And Exit ◽  
Wild Type

ABSTRACT Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV,srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had thesrf-3 amino acid sequence from E1 position 224 to 235 showed increases of ∼100-fold in infection and ∼250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGMexit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.

scholarly journals A Single Amino Acid Change within Antigenic Domain II of the Spike Protein of Bovine Coronavirus Confers Resistance to Virus Neutralization

2001 ◽  
Vol 8 (2) ◽  
pp. 297-302 ◽  
Author(s):  
Dongwan Yoo ◽  
Dirk Deregt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Bovine Coronavirus ◽  
Single Amino Acid Change ◽  
Group A ◽  
Escape Mutants ◽  
Group B

ABSTRACT The spike glycoprotein is a major neutralizing antigen of bovine coronavirus (BCV). Conformational neutralizing epitopes of group A and group B monoclonal antibodies (MAbs) have previously been mapped to two domains at amino acids 351 to 403 (domain I) and amino acids 517 to 621 (domain II). To further map antigenic sites, neutralization escape mutants of BCV were selected with a group A MAb which has both in vitro and in vivo virus-neutralizing ability. The escape mutants were demonstrated to be neutralization resistant to the selecting group A MAb and remained sensitive to neutralization by a group B MAb. In radioimmunoprecipitation assays, the spike proteins of neutralization escape mutants were shown to have lost their reactivities with the selecting group A MAb. Sequence analysis of the spike protein genes of the escape mutants identified a single nucleotide substitution of C to T at position 1583, resulting in the change of alanine to valine at amino acid position 528 (A528V). The mutation occurs in domain II and in a location which corresponds to the hypervariable region of the spike protein of the coronavirus mouse hepatitis virus. Experimental introduction of the A528V mutation into the wild-type spike protein resulted in the loss of MAb binding of the mutant protein, confirming that the single point mutation was responsible for the escape of BCV from immunological selective pressure.

scholarly journals A single-nucleotide change underlies the genetic assimilation of a plastic trait

2021 ◽  
Vol 7 (6) ◽  
pp. eabd9941
Author(s):  
Paul Vigne ◽  
Clotilde Gimond ◽  
Céline Ferrari ◽  
Anne Vielle ◽  
Johan Hallin ◽  
...  
Keyword(s):  
Evolutionary Biology ◽  
Single Point ◽  
Evolutionary Process ◽  
Resource Provisioning ◽  
Egg Laying ◽  
Genetic Assimilation ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Single Amino Acid Change ◽  
Plastic Trait

Genetic assimilation—the evolutionary process by which an environmentally induced phenotype is made constitutive—represents a fundamental concept in evolutionary biology. Thought to reflect adaptive phenotypic plasticity, matricidal hatching in nematodes is triggered by maternal nutrient deprivation to allow for protection or resource provisioning of offspring. Here, we report natural Caenorhabditis elegans populations harboring genetic variants expressing a derived state of near-constitutive matricidal hatching. These variants exhibit a single amino acid change (V530L) in KCNL-1, a small-conductance calcium-activated potassium channel subunit. This gain-of-function mutation causes matricidal hatching by strongly reducing the sensitivity to environmental stimuli triggering egg-laying. We show that reestablishing the canonical KCNL-1 protein in matricidal isolates is sufficient to restore canonical egg-laying. While highly deleterious in constant food environments, KCNL-1 V530L is maintained under fluctuating resource availability. A single point mutation can therefore underlie the genetic assimilation—by either genetic drift or selection—of an ancestrally plastic trait.

scholarly journals A single point mutation in a TssB/VipA homolog disrupts sheath formation in the type VI secretion system of Proteus mirabilis

2017 ◽  
Author(s):  
Christina C. Saak ◽  
Martha A. Zepeda-Rivera ◽  
Karine A. Gibbs
Keyword(s):  
Proteus Mirabilis ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Type Vi Secretion System ◽  
Self Identity ◽  
Type Vi Secretion ◽  
Self Recognition ◽  
Single Amino Acid Change ◽  
Sheath Formation

AbstractThe type VI secretion (T6S) system is a molecular device for the delivery of proteins from one cell into another. T6S function depends on the contractile sheath comprised of TssB/VipA and TssC/VipB proteins. We previously reported on a mutant variant of TssB that disrupts T6S-dependent export of the self-identity protein, IdsD, in the bacterium Proteus mirabilis. Here we determined the mechanism underlying that initial observation. We show that T6S-dependent export of multiple self-recognition proteins is abrogated in this mutant strain. We have mapped the mutation, which is a single amino acid change, to a region predicted to be involved in the formation of the TssB-TssC sheath. We have demonstrated that this mutation does indeed inhibit sheath formation, thereby explaining the global disruption of T6S activity. We propose that this mutation could be utilized as an important tool for studying functions and behaviors associated with T6S systems.

scholarly journals A single amino acid change in the E2 spike protein of a virulent strain of Semliki Forest virus attenuates pathogenicity

1994 ◽  
Vol 75 (3) ◽  
pp. 663-668 ◽  
Author(s):  
G. M. Glasgow ◽  
H. M. Killen ◽  
P. Liljestrom ◽  
B. J. Sheahan ◽  
G. J. Atkins
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Semliki Forest Virus ◽  
Virulent Strain ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Single Amino Acid Change

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells

2012 ◽  
Vol 87 (3) ◽  
pp. 1658-1663 ◽  
Author(s):  
Marco A. Díaz-Salinas ◽  
Pedro Romero ◽  
Rafaela Espinosa ◽  
Yasutaka Hoshino ◽  
Susana López ◽  
...  
Keyword(s):  
Surface Protein ◽  
Endocytic Pathway ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Cellular Receptors ◽  
Hypertonic Medium ◽  
Bovine Rotavirus ◽  
Single Amino Acid Change ◽  
Interfering Rna

ABSTRACTRotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface protein VP7 and the spike protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway.

scholarly journals Molecular basis of reduced or absent expression of decay-accelerating factor in Cromer blood group phenotypes

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1276-1282 ◽  
Author(s):  
DM Lublin ◽  
G Mallinson ◽  
J Poole ◽  
ME Reid ◽  
ES Thompson ◽  
...  
Keyword(s):  
Blood Group ◽  
Molecular Basis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Blood Group System ◽  
Single Nucleotide ◽  
Decay Accelerating Factor ◽  
Exon 2 ◽  
Nucleotide Deletion ◽  
Single Amino Acid Change

Abstract The human erythrocyte blood group system Cromer consists of high- incidence and low-incidence antigens that reside on decay-accelerating factor (DAF; CD55), a glycosyl-phosphatidylinositol-anchored membrane protein that regulates complement activation on cell surfaces. In the Cromer phenotypes Dr(a-) and Inab there is reduced or absent expression of DAF, respectively. This study investigated the molecular basis of the reduced DAF expression by polymerase chain reaction amplification of genomic DNA and RNA/cDNA obtained from Epstein-Barr virus- transformed lymphoblastoid cell lines. Sequence analysis of the Inab propositus showed a single nucleotide substitution in exon 2 of the DAF gene and at the corresponding position in the cDNA, G314-->A resulting in Trp53-->Stop. This truncation near the amino terminus explains the complete absence of surface DAF in the Inab phenotype. A similar analysis was performed for two Dr(a-) individuals, including KZ, who was previously reported to be Inab phenotype but is now shown by immunochemical and serologic methods to be Dr(a-) phenotype. A single nucleotide change was found in exon 5 of the DAF gene, C649-->T resulting in Ser165-->Leu, which we had previously shown to lead to loss of the Dra epitope. However, two species of cDNA were found, one encoding full-length DAF with the single amino acid change and the more abundant species having a 44-nucleotide deletion. The 44 nucleotide deletion includes the single polymorphic site, which creates a cryptic branch point in the Dr(a-) allele that leads to use of a downstream cryptic acceptor splice site. This shifts the reading frame and leads to a premature stop codon that precludes membrane anchoring. Thus, the single point mutation in the Dr(a-) phenotype results in a novel use of alternative splicing and provides a molecular explanation for both the antigenicity and the reduced DAF expression seen in this phenotype.

scholarly journals Novel Mutations That Control the Sphingolipid and Cholesterol Dependence of the Semliki Forest Virus Fusion Protein

2002 ◽  
Vol 76 (24) ◽  
pp. 12712-12722 ◽  
Author(s):  
Prodyot K. Chatterjee ◽  
Christina H. Eng ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Fusion Peptide ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Novel Mutations ◽  
E1 Protein ◽  
Target Membrane

ABSTRACT The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants, srf-4 and srf-5, selected for growth in cholesterol-depleted cells. Like the previously isolated srf-3 mutant (E1 proline 226 to serine), the phenotypes of the srf-4 and srf-5 mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like srf-3, srf-4 and srf-5 show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike srf-3, srf-4 and srf-5 showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both srf-4 and srf-5 formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the srf-3 mutant. All three srf mutations lie in the same domain of E1, but the srf-4 and srf-5 mutations are spatially separated from srf-3. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the srf-4 and srf-5 mutations identify novel regions of E1 that are distinct from the fusion peptide and srf-3 region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.

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