Site-directed mutagenesis of the VP2 gene of Chicken anemia virus affects virus replication, cytopathology and host-cell MHC class I expression

Chicken anemia virus (CAV) is an immunosuppressive pathogen of chickens. To further examine the role of viral protein 2 (VP2), which possesses dual-specificity protein phosphatase (DSP) activity, in viral cytopathogenicity and its influence on viral growth and virulence, an infectious genomic clone of CAV was subjected to site-directed mutagenesis. Substitution mutations C87R, R101G, K102D and H103Y were introduced into the DSP catalytic motif and R129G, Q131P, R/K/K150/151/152G/A/A, D/E161/162G/G, L163P, D169G and E186G into a region predicted to have a high degree of secondary structure. All mutant constructs were infectious, but their growth curves differed. The growth curve for mutant virus R/K/K150/151/152G/A/A was similar to that for wild-type virus, a second cluster of mutant viruses had an extended latent period and a third cluster of mutant viruses had extended latent and eclipse periods. All mutants had a reduced cytopathogenic effect in infected cells and VP3 was restricted to the cytoplasm. Mutation of the second basic residue (K102D) in the atypical DSP signature motif resulted in a marked reduction in virus replication efficiency, whereas mutation of the first basic residue (R101G) attenuated cytopathogenicity, but did not reduce replication efficiency. Expression of major histocompatibility complex (MHC) class I was markedly downregulated in cells infected with wild-type CAV, but not in those infected with mutants. This study further demonstrates the significance of VP2 in CAV replication and shows that specific mutations introduced into the gene encoding this protein can reduce virus replication, cytopathogenicity and downregulation of MHC I in infected cells.

Download Full-text

Zn-α2-glycoprotein, an MHC Class I-Related Glycoprotein Regulator of Adipose Tissues: Modification or Abrogation of Ligand Binding by Site-Directed Mutagenesis†

Biochemistry ◽

10.1021/bi051881v ◽

2006 ◽

Vol 45 (7) ◽

pp. 2035-2041 ◽

Cited By ~ 24

Author(s):

Lindsay C. McDermott ◽

June A. Freel ◽

Anthony P. West ◽

Pamela J. Bjorkman ◽

Malcolm W. Kennedy

Keyword(s):

Ligand Binding ◽

Mhc Class I ◽

Site Directed Mutagenesis ◽

Class I ◽

Directed Mutagenesis ◽

Adipose Tissues

Download Full-text

Conferring the Binding Properties of the Mouse MHC Class I-related Receptor, FcRn, onto the Human Ortholog by Sequential Rounds of Site-directed Mutagenesis

Journal of Molecular Biology ◽

10.1016/j.jmb.2004.11.014 ◽

2005 ◽

Vol 345 (5) ◽

pp. 1071-1081 ◽

Cited By ~ 48

Author(s):

Jinchun Zhou ◽

Fernando Mateos ◽

Raimund J. Ober ◽

E. Sally Ward

Keyword(s):

Mhc Class I ◽

Site Directed Mutagenesis ◽

Class I ◽

Directed Mutagenesis ◽

Binding Properties ◽

Human Ortholog

Download Full-text

Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2

Biochemical Journal ◽

10.1042/bj3490501 ◽

2000 ◽

Vol 349 (2) ◽

pp. 501-507 ◽

Cited By ~ 7

Author(s):

Hyun Min KOO ◽

Sung-Ook CHOI ◽

Hyun Mi KIM ◽

Yu Sam KIM

Keyword(s):

Bradyrhizobium Japonicum ◽

Catalytic Mechanism ◽

Sequence Similarity ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Basic Residue ◽

High Sequence Similarity ◽

Signature Sequences ◽

The Common

Malonamidase (MA) E2 was previously purified and characterized from Bradyrhizobium japonicum USDA 110. The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli. The recombinant MAE2 was purified to homogeneity from the transformed E. coli. The biochemical properties of the recombinant enzyme are essentially identical to those from wild-type B. japonicum. A database search showed that the MAE2 protein has a high sequence similarity with the common signature sequences of the amidase family. The only exception is that the aspartic residue in these signature sequences is replaced by a glutamine residue. In order to identify amino acid residues essential for enzyme activity, a series of site-directed mutagenesis studies and steady-state kinetic experiments were performed. Gln195, Ser199, Cys207 and Lys213 of the common signature sequences were selected for site-directed mutagenesis. Among the mutants, Q195D, Q195E and S199C showed less than 0.02% of the kcat value of the wild-type enzyme, and S199A, Q195L and Q195N exhibited no detectable catalytic activities. Mutants (K213L, K213R and K213H) obtained by replacement of the only conserved basic residue, Lys213, in the signature sequences, also displayed significant reductions (approx. 380-fold) in kcat value, whereas C207A kept full activity. These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln195, Ser199 and Lys213 are involved.

Download Full-text

Attenuation of chicken anemia virus by site-directed mutagenesis of VP2

Journal of General Virology ◽

10.1099/vir.0.82904-0 ◽

2007 ◽

Vol 88 (8) ◽

pp. 2168-2175 ◽

Cited By ~ 10

Author(s):

Michelle A. Peters ◽

Brendan S. Crabb ◽

Kelly A. Tivendale ◽

Glenn F. Browning

Keyword(s):

Bone Marrow ◽

Body Weight ◽

Site Directed Mutagenesis ◽

Chicken Anemia Virus ◽

Infection Model ◽

Wild Type ◽

Growth Depression ◽

Vp2 Gene ◽

Anemia Virus ◽

Type Infection

Chicken anemia virus (CAV) is a significant immunosuppressive pathogen of chickens, but relatively little is known about the effect of specific mutations on its virulence. In order to study the virulence of CAV, an infection model was developed in embryos. Significant growth depression, measured as a reduction in mean body weight, was found for wild-type CAV infection. Infection with wild-type CAV resulted in a significant reduction in thymic and splenic weights and consistently produced severe lesions in the thymus, spleen and bone marrow, as well as haemorrhages. CAVs mutated in the VP2 gene were infectious for embryos, but were highly attenuated with respect to growth depression and CAV-specific pathology. Relative to wild-type infection, viruses Mut C86R, Mut R101G, Mut H103Y, Mut R129G, Mut Q131P, Mut R/K/K150/151/152G/A/A, Mut D/E161/162G/G and Mut E186G were highly attenuated, and viruses Mut L163P and Mut D169G were moderately attenuated. Attenuation of the ability to produce lesions was found consistently for the thymus, spleen and bone marrow, thymic and splenic weights, and for CAV-induced haemorrhage. There was no growth depression associated with infection by the group of highly attenuated mutant viruses and a moderate reduction in mean body weight was only found for virus Mut L163P. These findings show that mutations in the VP2 gene can reduce the virulence of CAV and these mutant viruses may have value as vaccine candidates.

Download Full-text

Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation.

Journal of Experimental Medicine ◽

10.1084/jem.174.6.1629 ◽

1991 ◽

Vol 174 (6) ◽

pp. 1629-1637 ◽

Cited By ~ 69

Author(s):

J H Cox ◽

J R Bennink ◽

J W Yewdell

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Antigen Presentation ◽

Viral Proteins ◽

Genetically Engineered ◽

Class I ◽

Wild Type ◽

Histocompatibility Complex ◽

Infected Cells ◽

Lumenal Domain

The E3/19K glycoprotein of adenovirus functions to diminish recognition of adenovirus-infected cells by major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) by binding intracellular class I molecules and preventing them from reaching the plasma membrane. In the present study we have characterized the nature of the interaction between E3/19K and the H-2Kd (Kd) molecule. An E3/19K molecule genetically engineered to terminate six residues from its normal COOH terminus (delta E19), was found to associate with Kd in a manner indistinguishable from wild-type E3/19K. Unlike E3/19K, however, delta E19 was transported through the Golgi complex to the plasma membrane, where it could be detected biochemically and immunocytochemically using a monoclonal antibody specific for the lumenal domain of E3/19K. Importantly, delta E19 also differed from E3/19K in being unable to prevent the presentation of Kd-restricted viral proteins to CTLs. This is unlikely to be due to delta E19 having a lower avidity for Kd than E3/19K, since delta E19 was able to compete with E3/19K for Kd binding, both physically, and functionally in nullifying the E3/19K blockade of antigen presentation. These findings indicate that the ability of E3/19K to block antigen presentation is due solely to its ability to retain newly synthesized class I molecules in the endoplasmic reticulum.

Download Full-text

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽

10.1039/c5ra15220h ◽

2015 ◽

Vol 5 (93) ◽

pp. 76040-76047 ◽

Cited By ~ 11

Author(s):

Zhenya Chen ◽

Ye Li ◽

Yue Feng ◽

Liang Chen ◽

Qipeng Yuan

Keyword(s):

Active Site ◽

Simulation Analysis ◽

Docking Simulation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Proteus Vulgaris ◽

Molecular Docking Simulation ◽

Activity Enhancement ◽

First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

Download Full-text

Mutant Firefly Luciferase Enzymes Resistant to the Inhibition by Sodium Chloride

10.21203/rs.3.rs-243105/v1 ◽

2021 ◽

Author(s):

Satoshi Yawata ◽

Kenichi Noda ◽

Ai Shimomura ◽

Akio Kuroda

Keyword(s):

Sodium Chloride ◽

Double Mutant ◽

Luciferase Activity ◽

Firefly Luciferase ◽

Activity Level ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Original Activity ◽

Double Mutation

Abstract ObjectivesFirefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of firefly luciferase in the presence of sodium chloride.ResultsWe first obtained two mutant luciferase enzymes whose inhibition were alleviated and identified these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

Download Full-text

Infection with Listeria monocytogenes impairs sialic acid addition to host cell glycoproteins.

Journal of Experimental Medicine ◽

10.1084/jem.180.6.2137 ◽

1994 ◽

Vol 180 (6) ◽

pp. 2137-2145 ◽

Cited By ~ 15

Author(s):

M S Villanueva ◽

C J Beckers ◽

E G Pamer

Keyword(s):

Listeria Monocytogenes ◽

Sialic Acid ◽

Host Cell ◽

Mhc Class I ◽

Acid Content ◽

Class I ◽

Sialic Acid Content ◽

Infected Cells ◽

The Impact ◽

Glycosylation Defect

Listeria monocytogenes is a facultative intracellular bacterium that causes severe disease in neonates and immunocompromised adults. Although entry, multiplication, and locomotion of Listeria in the cytosol of infected cells are well described, the impact of such infection on the host cell is unknown. In this report, we investigate the effect of L. monocytogenes infection on MHC class I synthesis, processing, and intracellular trafficking. We show that L. monocytogenes infection interferes with normal processing of N-linked oligosaccharides on the major histocompatibility complex (MHC) class I heavy chain molecule, H-2Kd, resulting in a reduced sialic acid content. The glycosylation defect is more pronounced as the infection progresses and results from interference with the addition of sialic acid rather than its removal by a neuraminidase. The effect is found in two different cell lines and is not limited to MHC class I molecules since CD45, a surface glycoprotein, and LGP120, a lysosomal glycoprotein, are similarly affected by L. monocytogenes infection. The glycosylation defect is specific for infection by L. monocytogenes since neither Trypanosoma cruzi nor Yersinia enterocolitica, two other intracellular pathogens, reproduces the effect. The resultant hyposialylation of H-2Kd does not impair its surface expression in infected cells. Diminished sialic acid content of surface glycoproteins may enhance host-defense by increasing susceptibility to lysis and promoting clearance of Listeria-infected cells.

Download Full-text

Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj2790035 ◽

1991 ◽

Vol 279 (1) ◽

pp. 35-41 ◽

Cited By ~ 86

Author(s):

R Chambert ◽

M F Petit-Glatron

Keyword(s):

Bacillus Subtilis ◽

Intermediate Formation ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Chain Elongation ◽

Directed Mutagenesis ◽

Water Mixture ◽

Wild Type ◽

Wild Type Enzyme ◽

Sucrose Hydrolysis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

Download Full-text