Effect of basic amino acids and alkaline ph on uterine contractility in vitro

Amino acids have been implicated with virus infection and replication. Here, we demonstrate the effects of two basic amino acids, arginine and lysine, and their ester derivatives on infection of two enveloped viruses, SARS-CoV-2, and influenza A virus. We found that lysine and its ester derivative can efficiently block infection of both viruses in vitro. Furthermore, the arginine ester derivative caused a significant boost in virus infection. Studies on their mechanism of action revealed that the compounds potentially disturb virus uncoating rather than virus attachment and endosomal acidification. Our findings suggest that lysine supplementation and the reduction of arginine-rich food intake can be considered as prophylactic and therapeutic regimens against these viruses while also providing a paradigm for the development of broad-spectrum antivirals.

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Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the beta 6 position

Blood ◽

10.1182/blood.v84.4.1309.bloodjournal8441309 ◽

1994 ◽

Vol 84 (4) ◽

pp. 1309-1313

Author(s):

K Adachi ◽

CH Lai ◽

P Konitzer ◽

M Donahee ◽

A Campbell ◽

...

Keyword(s):

Amino Acids ◽

Expression System ◽

Basic Amino Acid ◽

Cellulose Acetate Electrophoresis ◽

Yeast Expression System ◽

Basic Amino Acids ◽

Beta 2 ◽

Hb Concentration ◽

Hb C

We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-- >Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)). rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis. The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C. The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb. However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K. Crystallization of native Hb C and both rHbs was inhibited by Hb F. These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C.

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NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2.

The Journal of Cell Biology ◽

10.1083/jcb.108.4.1237 ◽

1989 ◽

Vol 108 (4) ◽

pp. 1237-1243 ◽

Cited By ~ 55

Author(s):

E Szczesna-Skorupa ◽

B Kemper

Keyword(s):

Amino Acids ◽

Cytochrome P450 ◽

Proteolytic Processing ◽

Alkaline Solutions ◽

Hydrophobic Core ◽

Proteolytic Digestion ◽

Basic Amino Acids ◽

Microsomal Membranes ◽

Transfer Signal

Insertion of rabbit cytochrome P450IIC2 and its modified form, [2-lys,3-arg]P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. In contrast, when [2-lys,3-arg]-P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. After treatment with endoglycosidase H, the more slowly migrating species comigrated with [2-lys,3-arg]P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. Both the glycosylated and nonglycosylated forms of [2-lys,3-arg]P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. Similar results were obtained for a truncated species, [2-lys,3-arg]P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. In contrast to the proteolytic processing observed previously in a hybrid [2-lys,3-arg]P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of [2-lys,3-arg]P450IIC2 was observed in the presence of membranes. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from [2-lys,3-arg]P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.

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Investigation of exponential RNA amplification by the Qβ replicase complex

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314083971 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1602-C1602

Author(s):

Heidi Olesen ◽

Charlotte Knudsen ◽

Paulina Seweryn ◽

Ditlev Brodersen ◽

Zarina Kutlubaeva ◽

...

Keyword(s):

Amino Acids ◽

Nmr Spectroscopy ◽

Rna Virus ◽

Β Subunit ◽

Rna Amplification ◽

Nmr Studies ◽

Basic Amino Acids ◽

Rna Backbone

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent RNA polymerase (β-subunit) and the host-encoded translation elongation factors, EF-Ts and EF-Tu as well as the ribosomal protein S1. The Qβ replicase has an extraordinary ability to exponentially amplify RNA in vivo and in vitro. A prerequisite for this is release of product and template RNA as single strands that can serve as new templates in subsequent rounds of replication. The role of S1 in the Qβ replicase is not clear. Recently, S1 was found to promote release of single-stranded product in Qβ replicase–mediated RNA synthesis. We have undertaken NMR spectroscopy and crystallization trials to improve our understanding of distinct S1 domains in solution as well as the ribosome- and replicase-binding properties of S1. Expression of distinct S1 domains for NMR spectroscopy has been optimized by use of autoinduction and results in high yields of [13C15N]-labelled protein fragments. These have proven very suitable for NMR studies and spectra revealed both ordered and disordered regions in the protein. Studies are ongoing. The structure of the Qβ core complex was recently determined at 2.5Å resolution. Thus, co-crystallization of the Qβ core in complex with S1 domains was undertaken and different crystal forms were obtained. These initial crystals diffracted to 3.2Å resolution and data processing as well as further optimization of the crystals is ongoing. S1 is thought to bind the β-subunit close to a region lined with basic amino acids, which potentially could facilitate interactions with the template RNA backbone and split it from the product strand. We demonstrate that neutralization of these basic amino acids indeed decrease or abolish infectivity of the Qβ phage. However, only one mutation, R503A affects the exponential replication in vitro. Crystallization of the Qβ holo enzyme bound to a truncated legitimate RNA template will be the next step for investigation of the mechanism of exponential RNA amplification by Qβ replicase.

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Mutations of basic amino acids of NCp7 of human immunodeficiency virus type 1 affect RNA binding in vitro.

Journal of Virology ◽

10.1128/jvi.70.2.771-777.1996 ◽

1996 ◽

Vol 70 (2) ◽

pp. 771-777 ◽

Cited By ~ 63

Author(s):

E Schmalzbauer ◽

B Strack ◽

J Dannull ◽

S Guehmann ◽

K Moelling

Keyword(s):

Amino Acids ◽

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Rna Binding ◽

Basic Amino Acids ◽

Immunodeficiency Virus

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In vitro synthesis of West Nile virus proteins indicates that the amino-terminal segment of the NS3 protein contains the active centre of the protease which cleaves the viral polyprotein after multiple basic amino acids

Journal of General Virology ◽

10.1099/0022-1317-72-4-851 ◽

1991 ◽

Vol 72 (4) ◽

pp. 851-858 ◽

Cited By ~ 89

Author(s):

G. Wengler ◽

G. Czaya ◽

P. M. Farber ◽

J. H. Hegemann

Keyword(s):

Amino Acids ◽

West Nile Virus ◽

Terminal Segment ◽

Active Centre ◽

In Vitro Synthesis ◽

Basic Amino Acids ◽

Amino Terminal ◽

Ns3 Protein ◽

Viral Polyprotein

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Association of Influenza Virus Matrix Protein with Ribonucleoproteins

Journal of Virology ◽

10.1128/jvi.73.9.7467-7473.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7467-7473 ◽

Cited By ~ 105

Author(s):

Zhiping Ye ◽

Teresa Liu ◽

Daniel P. Offringa ◽

Jonathan McInnis ◽

Roland A. Levandowski

Keyword(s):

Amino Acids ◽

Zinc Finger ◽

Influenza A ◽

Matrix Protein ◽

Rna Binding ◽

Site Directed Mutagenesis ◽

Zinc Finger Motif ◽

Basic Amino Acids ◽

Binding Regions

ABSTRACT To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.

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Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the beta 6 position

Blood ◽

10.1182/blood.v84.4.1309.1309 ◽

1994 ◽

Vol 84 (4) ◽

pp. 1309-1313 ◽

Cited By ~ 3

Author(s):

K Adachi ◽

CH Lai ◽

P Konitzer ◽

M Donahee ◽

A Campbell ◽

...

Keyword(s):

Amino Acids ◽

Expression System ◽

Basic Amino Acid ◽

Cellulose Acetate Electrophoresis ◽

Yeast Expression System ◽

Basic Amino Acids ◽

Beta 2 ◽

Hb Concentration ◽

Hb C

Abstract We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-- >Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)). rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis. The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C. The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb. However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K. Crystallization of native Hb C and both rHbs was inhibited by Hb F. These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C.

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