scholarly journals Single Amino Acid Differences between Closely Related Reovirus T3D Lab Strains Alter Oncolytic Potency In Vitro and In Vivo

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Adil Mohamed ◽  
Derek R. Clements ◽  
Shashi A. Gujar ◽  
Patrick W. Lee ◽  
James R. Smiley ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Amino Acid ◽  
Cancer Therapy ◽  
Murine Model ◽  
Single Amino Acid ◽  
Mouse Tumor ◽  
Plaque Size ◽  
Oncolytic Activity

ABSTRACT Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL. Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD. Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL. Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies. IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

2020 ◽  
Author(s):  
Nicole M. Collette ◽  
Victoria H.I. Lao ◽  
Dina R. Weilhammer ◽  
Barbara Zingg ◽  
Shoshana D. Cohen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Model Systems ◽  
Single Amino Acid ◽  
Replication Rate ◽  
Adult Mice ◽  
Naturally Occurring ◽  
Viral Virulence

AbstractThe 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks.Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

scholarly journals Targeting Sphingolipids for Cancer Therapy

2021 ◽  
Vol 11 ◽  
Author(s):  
Osmel Companioni ◽  
Cristina Mir ◽  
Yoelsis Garcia-Mayea ◽  
Matilde E. LLeonart
Keyword(s):  
Clinical Trials ◽  
Cell Death ◽  
Cancer Therapy ◽  
Preclinical Studies ◽  
Hepatic Toxicity ◽  
Multiple Cancers ◽  
Extensive Class ◽  
Effective Drugs

Sphingolipids are an extensive class of lipids with different functions in the cell, ranging from proliferation to cell death. Sphingolipids are modified in multiple cancers and are responsible for tumor proliferation, progression, and metastasis. Several inhibitors or activators of sphingolipid signaling, such as fenretinide, safingol, ABC294640, ceramide nanoliposomes (CNLs), SKI-II, α-galactosylceramide, fingolimod, and sonepcizumab, have been described. The objective of this review was to analyze the results from preclinical and clinical trials of these drugs for the treatment of cancer. Sphingolipid-targeting drugs have been tested alone or in combination with chemotherapy, exhibiting antitumor activity alone and in synergism with chemotherapy in vitro and in vivo. As a consequence of treatments, the most frequent mechanism of cell death is apoptosis, followed by autophagy. Aslthough all these drugs have produced good results in preclinical studies of multiple cancers, the outcomes of clinical trials have not been similar. The most effective drugs are fenretinide and α-galactosylceramide (α-GalCer). In contrast, minor adverse effects restricted to a few subjects and hepatic toxicity have been observed in clinical trials of ABC294640 and safingol, respectively. In the case of CNLs, SKI-II, fingolimod and sonepcizumab there are some limitations and absence of enough clinical studies to demonstrate a benefit. The effectiveness or lack of a major therapeutic effect of sphingolipid modulation by some drugs as a cancer therapy and other aspects related to their mechanism of action are discussed in this review.

In-silico structural, functional and immunogenic characterization of Taenia solium TS14 protein

2017 ◽  
Author(s):  
Chitra Joshi ◽  
Siddharth Gautam
Keyword(s):  
Amino Acid ◽  
In Silico ◽  
Mutational Analysis ◽  
Solvent Accessibility ◽  
Taenia Solium ◽  
Secretory Protein ◽  
Single Amino Acid

TS14, a Cysticercosis cellulosae derived protein, has been exploited for immunodiagnosis of cysticercosis in humans and pigs. However, the information on structure, function, stability and immunogenicity of TS14 derived from different isolates is primarily lacking. The present study deals with in-silico characterization of six TS14 isolates. High thermostability and an isoelectric point of 9.41 were recorded. Based on N-terminal amino acid residues, high resistance to intracellular proteases with extended in-vivo and in-vitro half-lives was predicted. TS14 is foreseen as a secretory protein with a signal peptide and an extracellular localization. Structural analysis of TS14 exhibited the dominance of helices in the secondary structure (92% coverage) with majority of residues showing high and medium solvent accessibility. High lysine content and presence of multiple nucleotide binding sites in TS14 suggests interaction with RNA/DNA and a role in their metabolism. Immunogenic profiling predicted presence of four distinct B-cell epitopes. Mutational analysis based on the single amino acid substitutions among six TS14 isolates demonstrated minor variations in structural stability; however, all the substitutions were well tolerated. Moreover, all the isolates revealed almost identical immunogenic profile with an equivocal potential to elicit the antibody-mediated immune response.

scholarly journals A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus PropagationIn Vitrobut NotIn Vivo

2015 ◽  
Vol 89 (11) ◽  
pp. 6126-6130 ◽  
Author(s):  
Yuki Takamatsu ◽  
Kouichi Morita ◽  
Daisuke Hayasaka
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Single Amino Acid ◽  
Virus Propagation ◽  
Viral Loads ◽  
Unique Amino Acid

We identified a unique amino acid of NS2A113, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagationin vitromay not reflect the level of virus neuroinvasivenessin vivo.

scholarly journals Fine Molecular Tuning of Chimeric Antigen Receptors through Hinge Length Optimization

2020 ◽  
Author(s):  
Scott McComb ◽  
Tina Nguyen ◽  
Kevin A. Henry ◽  
Darin Bloemberg ◽  
Susanne Maclean ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Specific Activity ◽  
Single Amino Acid ◽  
Single Domain Antibody ◽  
Normal Tissues ◽  
Epidermal Growth

AbstractBackgroundChimeric antigen receptor (CAR) technology has revolutionized the treatment of B-cell malignancies and steady progress is being made towards CAR-immunotherapies for solid tumours. In the context of CARs targeting antigens which are commonly overexpressed in cancer but also expressed at lower levels in normal tissues, such as epidermal growth factor family receptors EGFR or HER2, it is imperative that any targeting strategy consider the potential for on-target off-tumour toxicity. Molecular optimization of the various protein domains of CARs can be used to increase the tumour selectivity.MethodHerein, we utilize high-throughput CAR screening to identify a novel camelid single-domain antibody CAR (sdCAR) targeting human epidermal growth factor (EGFR) with high EGFR-specific activity. To further optimize the target selectivity of this EGFR-sdCAR, we performed progressive N-terminal single amino acid truncations of an extended human CD8 hinge domain [(G4S)3GG-45CD8h] to improve selectivity for EGFR-overexpressing cells. We also make direct comparison of varying hinge domains in scFv-based CARs targeting EGFR-family tumour associated antigens EGFRvIII and HER2.ResultsThrough comparison of various hinge-truncated scFv- and sdAb-based CARs, we show that the CAR hinge/spacer domain plays varying roles in modifying CAR signaling depending upon target epitope location. For membrane-proximal epitopes, hinge truncation by even a single amino acid resulted in fine control of CAR signaling strength. Hinge-modified CARs showed consistent and predictable signaling in Jurkat-CAR cells and primary human CAR-T cells in vitro and in vivo.ConclusionsOverall, these results indicate that membrane-proximal epitope targeting CARs can be optimized through hinge length tuning for improved target selectivity and therapeutic function. Graphical Abstract

scholarly journals Single Amino Acid Mutations Affect Zika Virus Replication In Vitro and Virulence In Vivo

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1295
Author(s):  
Nicole M. Collette ◽  
Victoria H. I. Lao ◽  
Dina R. Weilhammer ◽  
Barbara Zingg ◽  
Shoshana D. Cohen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Model Systems ◽  
Single Amino Acid ◽  
Replication Rate ◽  
Adult Mice ◽  
Naturally Occurring ◽  
Viral Virulence

The 2014–2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

scholarly journals A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Sujit K. Mohanty ◽  
Bryan Donnelly ◽  
Phylicia Dupree ◽  
Inna Lobeck ◽  
Sarah Mowery ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biliary Atresia ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Wild Type ◽  
Neonatal Mice ◽  
Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

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