scholarly journals Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

2006 ◽  
Vol 80 (11) ◽  
pp. 5413-5422 ◽  
Author(s):  
Aleksei Lulla ◽  
Valeria Lulla ◽  
Kairit Tints ◽  
Tero Ahola ◽  
Andres Merits
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Cleavage Site ◽  
Semliki Forest Virus ◽  
Nonstructural Protein ◽  
Cleavage Sites ◽  
Vitro Assay ◽  
Virus Life Cycle ◽  
Cleavage Efficiency

ABSTRACT The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1′ had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1′, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.

scholarly journals Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1092
Author(s):  
János András Mótyán ◽  
Márió Miczi ◽  
Stephen Oroszlan ◽  
József Tőzsér
Keyword(s):  
Amino Acid ◽  
Zinc Finger ◽  
Cleavage Site ◽  
Potential Role ◽  
Binding Mode ◽  
Interaction Energies ◽  
Vitro Assay ◽  
Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

scholarly journals Predicted Coronavirus Nsp5 Protease Cleavage Sites in the Human Proteome: A Resource for SARS-CoV-2 Research

2021 ◽  
Author(s):  
Benjamin M Scott ◽  
Vincent Lacasse ◽  
Ditte G Blom ◽  
Peter D Tonner ◽  
Nikolaj S Blom
Keyword(s):  
Protein Interactions ◽  
Cleavage Site ◽  
Nonstructural Protein ◽  
Data Bank ◽  
Molecular Pathways ◽  
Cleavage Sites ◽  
Host Proteins ◽  
Structure Information ◽  
Human Proteins

Background: The coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach. Results: Using a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis. Conclusions: This study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of SARS-CoV-2 infection.

scholarly journals Functional and Genetic Studies of the Substrate Specificity of Coronavirus Infectious Bronchitis Virus 3C-Like Proteinase

2010 ◽  
Vol 84 (14) ◽  
pp. 7325-7336 ◽  
Author(s):  
Shouguo Fang ◽  
Hongyuan Shen ◽  
Jibin Wang ◽  
Felicia P. L. Tay ◽  
Ding Xiang Liu
Keyword(s):  
Infectious Bronchitis Virus ◽  
Nonstructural Protein ◽  
Cleavage Sites ◽  
Nonstructural Proteins ◽  
Genetic Studies ◽  
Subgenomic Rna ◽  
Infectious Bronchitis ◽  
Growth Defects ◽  
Cleavage Efficiency

ABSTRACT Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.

scholarly journals H5N2 Avian Influenza Outbreak in Texas in 2004: the First Highly Pathogenic Strain in the United States in 20 Years?

2005 ◽  
Vol 79 (17) ◽  
pp. 11412-11421 ◽  
Author(s):  
Chang-Won Lee ◽  
David E. Swayne ◽  
Jose A. Linares ◽  
Dennis A. Senne ◽  
David L. Suarez
Keyword(s):  
United States ◽  
Amino Acid ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Basic Amino Acid ◽  
The United States ◽  
Influenza Viruses ◽  
Single Point Mutation ◽  
Highly Pathogenic

ABSTRACT In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.

scholarly journals A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1862-1862
Author(s):  
Gregory J. Cost ◽  
Morayma Temoche-Diaz ◽  
Janet Mei ◽  
Cristina N. Butterfield ◽  
Christopher T. Brown ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Attractive Alternative ◽  
Vitro Assay ◽  
Crispr System ◽  
Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

In Vitro Assay for Predicting Protein Efficiency Ratio as Measured by Rat Bioassay: Collaborative Study

1982 ◽  
Vol 65 (4) ◽  
pp. 798-809 ◽  
Author(s):  
Lowell D Satterlee ◽  
James G Kendrick ◽  
Henry F Marshall ◽  
Duane K Jewell ◽  
Rida A Ali ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Efficiency Ratio ◽  
Muscle Protein ◽  
Collaborative Study ◽  
Protein Digestibility ◽  
Efficiency Ratio ◽  
Breakfast Cereal ◽  
Vitro Assay ◽  
Food Ingredients

Abstract Seven laboratories collaborated in testing the calculated protein efficiency ratio (C-PER and DC-PER). The collaborative study required each laboratory to analyze 6 foods and a control protein (ANRC casein) for in vitro apparent protein digestibility, amino acid composition, and PER via rat bioassay. The 6 foods or food ingredients tested were nonfat dry milk, cooked chicken muscle, protein-fortified dry breakfast cereal, textured soy protein, oat-based dry breakfast cereal, and durum wheat flour. Data obtained from the study were analyzed statistically for the intralaboratory variation for each method of analysis (i.e., amino acid analysis, PER, etc.). The ability of the C-PER to rapidly predict rat PER was also measured. The C-PER and DC-PER methods were adopted official first action.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

scholarly journals Nutritional availability of methionine, lysine and tryptophan in fish meals, as assessed with biological, microbiological and dye-binding assay procedures

1985 ◽  
Vol 53 (3) ◽  
pp. 575-586 ◽  
Author(s):  
D. Hewitt ◽  
J. E. Ford
Keyword(s):  
Amino Acid ◽  
Tetrahymena Pyriformis ◽  
Poor Quality ◽  
Analysis Procedure ◽  
Chick Growth ◽  
Vitro Assay ◽  
Similar Treatment ◽  
Dye Binding ◽  
Available Lysine

1. In vitro assay procedures were applied in the measurement of available amino acids in a selection of fish meals representing good- and poor-quality product. Results were assessed by comparing them with results from chick-growth assays.2. Available methionine and tryptophan were assayed microbiologically with Streptococcus zymogenes, after predigestion of the test samples with papain or pronase. Results for methionine were correlated with chick-growth assays (r 0.86 for papain, 0.91 for pronase; P < 0.01). Compared with the chick assays, papain tended to give lower, and pronase higher, results. Finer milling of the test samples did not influence the pronase values.3. Results for available tryptophan were also correlated with chick-growth assays (r 0.95 for papain, 0.96 for pronase; P < 0.001). Compared with the chick values, papain gave markedly lower results and pronasem marginally higher ones. Finer milling of the test samples increased the papain values by about 50% but had no effect with pronase.4. Available lysine was assayed microbiologically with Tetrahymena pyriformis and with a dye-binding procedure (DBL). The results correlated with the chick-growth assays (r 0.99 for DBL, P < 0.001; 0.85 for Tetrahymena, P < 0.01) but both methods overrated the poorer-quality samples.5. True nitrogen digestibilities and amino acid digestibilities were determined with chickens by the 'ileal analysis' procedure: the amino acid digestibilities were significantly higher and similar to the corresponding availabilities as measured in chick-growth assays.6. Ball milling a poor-quality fish meal caused a marked fall in its N digestibility, whereas similar treatment of a good-quality meal caused a slight increase. An explanation for this finding is proposed.7. Strep. zymogenes assays following pronase digestion of the test samples gave precise and acceptably accurate measures of the biologically available methionine and tryptophan in the test samples. For available lysine, Tetrahymena and DBL values for the poorer-quality samples were notably higher than the chick-growth assay; possible reasons for this are discussed. The ileal analysis procedure underestimated true N digestibility.

scholarly journals QUANTITATIVE EFFECTS OF NUTRITIONAL ESSENTIAL AMINO ACID DEFICIENCY UPON IMMUNE RESPONSES TO TUMORS IN MICE

1973 ◽  
Vol 137 (1) ◽  
pp. 1-9 ◽  
Author(s):  
David G. Jose ◽  
Robert A. Good
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Immune Responses ◽  
Tumor Growth ◽  
Essential Amino Acid ◽  
Cell Mediated Immunity ◽  
Host Animal ◽  
Vitro Assay ◽  
Cytotoxic Cell

Mice were fed diets deficient in a single essential amino acid, and the primary immune responses to inoculation of allogenic tumor cells was measured by in vitro assay of cellular immunity. Moderate reduction of the amino acids phenylalanine-tyrosine, valine, threonine, methionine-cystine, isoleucine, and tryptophane in the diet produced profound depression of hemagglutinating and blocking antibody responses, although cytotoxic cell-mediated immunity remained intact. These diets had previously been shown to result in a selective depression of tumor growth in mice. Limitation of the amino acids arginine, histidine, and lysine in the diets gave rise to only slight depression of the immune responses. These diets had previously been shown to produce a proportional decrease in both tumor growth and host body weight. Moderate leucine restriction resulted in a paradoxical depression of cytotoxic cell-mediated immunity with little effect on serum blocking activity. Slight increases had previously been noted in the weight of tumors in mice fed leucine-restricted diets. Deficiency or imbalance of essential amino acids in the diet may produce profound depression of immune responses and apparent, marked changes in the immune resistance of the host animal to tumors.

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