scholarly journals Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine.

1996 ◽  
Vol 40 (9) ◽  
pp. 1983-1987 ◽  
Author(s):  
Y Q Zhu ◽  
K M Remington ◽  
T W North
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Cultured Cells ◽  
Single Point ◽  
Site Directed Mutagenesis ◽  
Single Point Mutation ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Phosphonoformic Acid ◽  
Immunodeficiency Virus

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis.

scholarly journals Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

1996 ◽  
Vol 40 (4) ◽  
pp. 953-957 ◽  
Author(s):  
H K Medlin ◽  
Y Q Zhu ◽  
K M Remington ◽  
T R Phillips ◽  
T W North
Keyword(s):  
Reverse Transcriptase ◽  
Feline Immunodeficiency Virus ◽  
Cultured Cells ◽  
Base Change ◽  
Site Directed Mutagenesis ◽  
Phosphonoformic Acid ◽  
Immunodeficiency Virus ◽  
Single Base Change ◽  
Continuous Presence

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

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 Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c

Pathogens ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 157 ◽  
Author(s):  
Johannes Scholz ◽  
Christine Bächlein ◽  
Ashish K. Gadicherla ◽  
Alexander Falkenhagen ◽  
Simon H. Tausch ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis E Virus ◽  
Reverse Genetics ◽  
Single Point ◽  
Hepatitis E ◽  
Site Directed Mutagenesis ◽  
Additional Restriction ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Directed Mutagenesis

The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.

scholarly journals Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

2000 ◽  
Vol 350 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Ana M. PAJOR ◽  
Esther S. KAHN ◽  
Rama GANGULA
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
In The Wild ◽  
Cationic Amino Acids

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain

1991 ◽  
Vol 99 (2) ◽  
pp. 351-362 ◽  
Author(s):  
M. Hatzfeld ◽  
K. Weber
Keyword(s):  
Intermediate Filament ◽  
Single Point ◽  
Consensus Sequence ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Wild Type ◽  
Filament Assembly ◽  
Elongation Process

All known intermediate filament (IF) proteins display -8 -4 -1 a consensus sequence TYRKLLEGE at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position -8 was changed to alanine in keratin K8 (K8Y----A-8) and keratin K18 (K18Y----A-8) and leucine at position -4 was changed to glycine in keratin K18 (K18L----G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins. In standard filament buffer (50 mM Tris-HCl, pH7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH7.5) mutant keratins K8Y----A-8 plus K18Y----A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L----G-4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly.

scholarly journals Mutations at embB Codon 306 Are an Important Molecular Indicator of Ethambutol Resistance in Mycobacterium tuberculosis

2008 ◽  
Vol 53 (3) ◽  
pp. 1061-1066 ◽  
Author(s):  
Angela M. Starks ◽  
Aysel Gumusboga ◽  
Bonnie B. Plikaytis ◽  
Thomas M. Shinnick ◽  
James E. Posey
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Allele ◽  
Single Point ◽  
Single Point Mutation ◽  
Missense Mutations ◽  
Wild Type ◽  
Molecular Tests ◽  
Resistant Mutants ◽  
Spontaneous Mutants

ABSTRACT Ethambutol resistance in clinical Mycobacterium tuberculosis isolates is associated primarily with missense mutations in the embB gene. However, recent reports have described the presence of embB mutations, especially those at embB codon 306, in isolates susceptible to ethambutol. To clarify the role of embB mutations in ethambutol resistance, we sequenced the ethambutol resistance-determining region in spontaneous ethambutol-resistant mutants. In our study, 66% of spontaneous mutants contained a single point mutation in embB, with 55% of these occurring at embB 306. The MIC of ethambutol for spontaneous mutants was increased two- to eightfold relative to the pansusceptible M. tuberculosis strains from which the mutants were generated. To further characterize the role of embB 306 mutations, we directly introduced mutant alleles, embB(M306V) or embB(M306I), into pansusceptible M. tuberculosis strains and conversely reverted mutant alleles in spontaneous ethambutol-resistant mutants back to those of the wild type via allelic exchange using specialized linkage transduction. We determined that the MIC of ethambutol was reduced fourfold for three of the four spontaneous ethambutol-resistant embB 306 mutants when the mutant allele was replaced with the wild-type embB allele. The MIC for one of the spontaneous mutants genetically reverted to wild-type embB was reduced by only twofold. When the wild-type embB allele was converted to the mutant allele embB(M306V), the ethambutol MIC was increased fourfold, and when the allele was changed to M306I, the ethambutol MIC increased twofold. Our data indicate that embB 306 mutations are sufficient to confer ethambutol resistance, and detection of these mutations should be considered in the development of rapid molecular tests.

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