Characterization of Poliovirus Variants Selected for Resistance to the Antiviral Compound V-073

ABSTRACTV-073, a small-molecule capsid inhibitor originally developed for nonpolio enterovirus indications is considerably more potent against polioviruses. All poliovirus isolates tested to date (n= 45), including wild, vaccine, vaccine-derived, and laboratory strains, are susceptible to the antiviral capsid inhibitor V-073. We grew poliovirus in the presence of V-073 to allow for the identification of variants with reduced susceptibility to the drug. Sequence analysis of 160 independent resistant variants (80 isolates of poliovirus type 1, 40 isolates each of types 2 and 3) established that V-073 resistance involved a single amino acid change in either of two virus capsid proteins, VP1 (67 of 160 [42%]) or VP3 (93 of 160 [58%]). In resistant variants with a VP1 change, the majority (53 of 67 [79%]) exhibited a substitution of isoleucine at position 194 (equivalent position 192 in type 3) with either methionine or phenylalanine. Of those with a VP3 change, alanine at position 24 was replaced with valine in all variants (n= 93). The resistance phenotype was relatively stable upon passage of viruses in cell culture in the absence of drug. Single-step growth studies showed no substantial differences between drug-resistant variants and the virus stocks from which they were derived, while the resistant viruses were generally more thermally labile than the corresponding drug-susceptible parental viruses. These studies provide a foundation from which to build a greater understanding of resistance to antiviral compound V-073.

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The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells

Journal of Virology ◽

10.1128/jvi.02086-12 ◽

2012 ◽

Vol 87 (3) ◽

pp. 1658-1663 ◽

Cited By ~ 31

Author(s):

Marco A. Díaz-Salinas ◽

Pedro Romero ◽

Rafaela Espinosa ◽

Yasutaka Hoshino ◽

Susana López ◽

...

Keyword(s):

Surface Protein ◽

Endocytic Pathway ◽

Spike Protein ◽

Single Amino Acid ◽

Cellular Receptors ◽

Hypertonic Medium ◽

Bovine Rotavirus ◽

Single Amino Acid Change ◽

Interfering Rna

ABSTRACTRotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface protein VP7 and the spike protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway.

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Position β57 of I-Ag7 controls early anti-insulin responses in NOD mice, linking an MHC susceptibility allele to type 1 diabetes onset

Science Immunology ◽

10.1126/sciimmunol.aaw6329 ◽

2019 ◽

Vol 4 (38) ◽

pp. eaaw6329 ◽

Cited By ~ 9

Author(s):

Louis Gioia ◽

Marie Holt ◽

Anne Costanzo ◽

Siddhartha Sharma ◽

Brian Abe ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

Disease Onset ◽

Insulin Response ◽

Nod Mice ◽

Class Ii ◽

Single Amino Acid ◽

Specific Gene ◽

Single Amino Acid Change

The class II region of the major histocompatibility complex (MHC) locus is the main contributor to the genetic susceptibility to type 1 diabetes (T1D). The loss of an aspartic acid at position 57 of diabetogenic HLA-DQβ chains supports this association; this single amino acid change influences how TCRs recognize peptides in the context of HLA-DQ8 and I-Ag7 using a mechanism termed the P9 switch. Here, we built register-specific insulin peptide MHC tetramers to examine CD4+ T cell responses to Ins12–20 and Ins13–21 peptides during the early prediabetic phase of disease in nonobese diabetic (NOD) mice. A single-cell analysis of anti-insulin CD4+ T cells performed in 6- and 12-week-old NOD mice revealed tissue-specific gene expression signatures. TCR signaling and clonal expansion were found only in the islets of Langerhans and produced either classical TH1 differentiation or an unusual Treg phenotype, independent of TCR usage. The early phase of the anti-insulin response was dominated by T cells specific for Ins12–20, the register that supports a P9 switch mode of recognition. The presence of the P9 switch was demonstrated by TCR sequencing, reexpression, mutagenesis, and functional testing of TCRαβ pairs in vitro. Genetic correction of the I-Aβ57 mutation in NOD mice resulted in the disappearance of D/E residues in the CDR3β of anti-Ins12–20 T cells. These results provide a mechanistic molecular explanation that links the characteristic MHC class II polymorphism of T1D with the recognition of islet autoantigens and disease onset.

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Characterization of cDNAs encoding isoforms of hamster 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0200099 ◽

1998 ◽

Vol 20 (1) ◽

pp. 99-110 ◽

Cited By ~ 9

Author(s):

FM Rogerson ◽

J Courtemanche ◽

A Fleury ◽

JG LeHoux ◽

JI Mason ◽

...

Keyword(s):

Hydroxysteroid Dehydrogenase ◽

Cdna Libraries ◽

Sexually Dimorphic ◽

High Affinity ◽

Liver And Kidney ◽

Low Levels ◽

Type 3

Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.

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Determinants of Syncytium Formation in Microglia by Human Immunodeficiency Virus Type 1: Role of the V1/V2 Domains

Journal of Virology ◽

10.1128/jvi.74.2.693-701.2000 ◽

2000 ◽

Vol 74 (2) ◽

pp. 693-701 ◽

Cited By ~ 35

Author(s):

Joseph T. C. Shieh ◽

Julio Martín ◽

Gordon Baltuch ◽

Michael H. Malim ◽

Francisco González-Scarano

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Syncytium Formation ◽

Single Amino Acid ◽

Single Amino Acid Change ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Microglia are the main reservoir for human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS), and multinucleated giant cells, the result of fusion of HIV-1-infected microglia and brain macrophages, are the neuropathologic hallmark of HIV dementia. One potential explanation for the formation of syncytia is viral adaptation for these CD4+ CNS cells. HIV-1BORI-15, a virus adapted to growth in microglia by sequential passage in vitro, mediates high levels of fusion and replicates more efficiently in microglia and monocyte-derived-macrophages than its unpassaged parent (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. Gonzalez-Scarano, J. Virol. 70:7654–7662, 1996). Since the interaction between the viral envelope glycoprotein and CD4 and the chemokine receptor mediates fusion and plays a key role in tropism, we have analyzed the HIV-1BORI-15 env as a fusogen and in recombinant and pseudotyped viruses. Its syncytium-forming phenotype is not the result of a switch in coreceptor use but rather of the HIV-1BORI-15envelope-mediated fusion of CD4+CCR5+ cells with greater efficiency than that of its parental strain, either by itself or in the context of a recombinant virus. Genetic analysis indicated that the syncytium-forming phenotype was due to four discrete amino acid differences in V1/V2, with a single-amino-acid change between the parent and the adapted virus (E153G) responsible for the majority of the effect. Additionally, HIV-1BORI-15 env-pseudotyped viruses were less sensitive to decreases in the levels of CD4 on transfected 293T cells, leading to the hypothesis that the differences in V1/V2 alter the interaction between this envelope and CD4 or CCR5, or both. In sum, the characterization of the envelope of HIV-1BORI-15, a highly fusogenic glycoprotein with genetic determinants in V1/V2, may lead to a better understanding of the relationship between HIV replication and syncytium formation in the CNS and of the importance of this region of gp120 in the interaction with CD4 and CCR5.

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Novel LinA Type 3 δ-Hexachlorocyclohexane Dehydrochlorinase

Applied and Environmental Microbiology ◽

10.1128/aem.01683-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7553-7559 ◽

Cited By ~ 4

Author(s):

Nidhi Shrivastava ◽

Zbynek Prokop ◽

Ashwani Kumar

Keyword(s):

Amino Acid ◽

Primary Structure ◽

Degradation Pathway ◽

Amino Acid Residues ◽

Hch Isomers ◽

New Variant ◽

Type 3

ABSTRACTLinA is the first enzyme of the microbial degradation pathway of a chlorinated insecticide, hexachlorocyclohexane (HCH), and mediates the dehydrochlorination of α-, γ-, and δ-HCH. Its two variants, LinA type 1 and LinA type 2, which differ at 10 out of 156 amino acid residues, have been described. Their activities for the metabolism of different HCH isomers differ considerably but overall are high for γ-HCH, moderate for α-HCH, low for δ-HCH, and lacking for β-HCH. Here, we describe the characterization of a new variant of this enzyme, LinA type 3, whose gene was identified from the metagenome of an HCH-contaminated soil sample. Its deduced primary structure in the region spanning amino acid residues 1 to 147 of the protein exhibits 17 and 12 differences from LinA type 1 and LinA type 2, respectively. In addition, the residues GIHFAPS, present at the region spanning residues 148 to 154 in both LinA type 1 and LinA type 2, are deleted in LinA type 3.The activity of LinA type 3 for the metabolism of δ-HCH is several orders of magnitude higher than that of LinA type 1 or LinA type 2 and can be useful for improvement of the metabolism of δ-HCH.

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Characterization of Abnormal Thymidine Kinases Induced by Drug-resistant Strains of Herpes Simplex Virus Type 1

Journal of General Virology ◽

10.1099/0022-1317-64-3-523 ◽

1983 ◽

Vol 64 (3) ◽

pp. 523-532 ◽

Cited By ~ 57

Author(s):

B. A. Larder ◽

Y.-C. Cheng ◽

G. Darby

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Drug Resistant ◽

Resistant Strains ◽

Simplex Virus ◽

Drug Resistant Strains

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Single Step Growth and Characterization of Zinc Oxide, Tin Oxide, and Composite (ZnxSn1−xOy) Nanoplate and Nanocolumn Electrodes

Journal of the American Ceramic Society ◽

10.1111/j.1551-2916.2011.04525.x ◽

2011 ◽

Vol 94 (10) ◽

pp. 3540-3546 ◽

Cited By ~ 16

Author(s):

Ruvini Dharmadasa ◽

Asif A. Tahir ◽

K. G. Upul Wijayantha

Keyword(s):

Zinc Oxide ◽

Tin Oxide ◽

Single Step ◽

Step Growth

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Triazene derivatives of (1,x)-diazacycloalkanes — Part IX. Synthesis and characterization of a series of 1,4-di[2-aryl-1-diazenyl]-2,6-dimethylpiperazines

Canadian Journal of Chemistry ◽

10.1139/v10-008 ◽

2010 ◽

Vol 88 (4) ◽

pp. 344-351 ◽

Cited By ~ 3

Author(s):

Naomi Hunter ◽

Keith Vaughan

Keyword(s):

Diazonium Salt ◽

Mass Measurement ◽

2D Nmr ◽

Piperazine Ring ◽

Ir And Nmr Spectroscopy ◽

Methylene Groups ◽

Type 3 ◽

Derivatives Of

A series of 1,4-di-[2-aryl-1-diazenyl]-2,6-dimethylpiperazines (5a–5l), have been synthesized by the reaction of 2,6-dimethylpiperazine with 2 equiv. of the appropriate diazonium salt. The products have been characterized by IR and NMR spectroscopy, and the molecular composition has been verified by high-resolution EI mass spectrometry with accurate mass measurement of the molecular ion. The presence of stereocenters at C2 and C6 of the piperazine ring in the bis-triazene 5 creates two unique pairs of diastereotopic protons in the methylene groups at positions 3 and 5 of the piperazine ring, as evidenced by the complexity of the NMR spectra, which nevertheless can be fully assigned in most cases. The assignment of the proton and carbon signals in the 1,4-di-[2-aryl-1-diazenyl]-2,6-dimethylpiperazines has been aided by the use of 2D NMR HSQC spectroscopy. These results compare favorably with assignments of proton and carbon signals reported previously for triazenes of type 1 and bis-triazenes of type 3.

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Characterization of an infectious cDNA copy of the genome of a naturally occurring, avirulent coxsackievirus B3 clinical isolate

Journal of General Virology ◽

10.1099/vir.0.80424-0 ◽

2005 ◽

Vol 86 (1) ◽

pp. 197-210 ◽

Cited By ~ 26

Author(s):

C.-K. Lee ◽

K. Kono ◽

E. Haas ◽

K.-S. Kim ◽

K. M. Drescher ◽

...

Keyword(s):

Avirulent Strain ◽

Cdna Copy ◽

Naturally Occurring ◽

Pathogenic Virus ◽

Phenotype Comparison ◽

Group B ◽

Type 3 ◽

Virus Strains

Group B coxsackieviruses (CVB) cause numerous diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes. To date, infectious cDNA copies of CVB type 3 (CVB3) genomes have all been derived from pathogenic virus strains. An infectious cDNA copy of the well-characterized, non-pathogenic CVB3 strain GA genome was cloned in order to facilitate mapping of the CVB genes that influence expression of a virulence phenotype. Comparison of the sequence of the parental CVB3/GA population, derived by direct RT-PCR-mediated sequence analysis, to that of the infectious CVB3/GA progeny genome demonstrated that an authentic copy was cloned; numerous differences were observed in coding and non-coding sequences relative to other CVB3 strains. Progeny CVB3/GA replicated similarly to the parental strain in three different cell cultures and was avirulent when inoculated into mice, causing neither pancreatitis nor myocarditis. Inoculation of mice with CVB3/GA protected mice completely against myocarditis and pancreatitis induced by cardiovirulent CVB3 challenge. The secondary structure predicted for the CVB3/GA domain II, a region within the 5′ non-translated region that is implicated as a key site affecting the expression of a cardiovirulent phenotype, differs from those predicted for cardiovirulent and pancreovirulent CVB3 strains. This is the first report characterizing a cloned CVB3 genome from an avirulent strain.

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