scholarly journals Deletion, insertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patients

2009 ◽  
Vol 3 (07) ◽  
pp. 531-538 ◽  
Author(s):  
Héctor Rafael Rangel ◽  
Domingo Garzaro ◽  
Anny Karely Rodríguez ◽  
Alvaro Hernán Ramírez ◽  
Gladys Ameli ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Viral Gene ◽  
Background Variable ◽  
Wild Type ◽  
Gene Defects ◽  
Hiv 1 ◽  
Variable Progression

Background: Variable progression towards AIDS has been described and has been related to viral and host factors. Around 10% of the HIV-1 infected patients are slow progressors (SP), not presenting with AIDS disease signs even after more than 10 years of infection. Viral gene defects have been associated with the disease progression but more studies are still needed. Methodology: The sequence of vif and nef were analyzed for HIV-1 infecting 14 SP and 46 normal progressors (NP) patients. Results: Co-circulation of a strain carrying vif deleted gene with the wild type strain was detected in an SP patient with more than 10 years of infection. Other mutations (insertion in aa 63 in one strain, two premature stop codons in another one) were found in viruses infecting two other patients. Except for the SP8 strain, which exhibited a premature stop codon in nef, no gross deletions or insertions were observed in nef genes of both NPs and SPs strains analyzed. Conclusions: Different kind of mutation: deletion, insertion and stop codon, were detected in 3/14 samples from SP, with co-circulation of a 195 bp vif deletion virus with a wild type in one of these patients. Although vif defects do not seem to be a frequent feature in SPs, this study illustrates the importance of analysing this gene, in addition to the multiple factors associated with the long-term non progression to AIDS.

scholarly journals Effective Suppression of Human Immunodeficiency Virus Type 1 through a Combination of Short- or Long-Hairpin RNAs Targeting Essential Sequences for Retroviral Integration

2006 ◽  
Vol 80 (15) ◽  
pp. 7658-7666 ◽  
Author(s):  
Hironori Nishitsuji ◽  
Michinori Kohara ◽  
Mari Kannagi ◽  
Takao Masuda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Attachment Site ◽  
High Dose ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Small interfering RNA (siRNA) could provide a new therapeutic approach to treating human immunodeficiency virus type 1 (HIV-1) infection. For long-term suppression of HIV-1, emergence of siRNA escape variants must be controlled. Here, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNA) corresponding to conserved target sequences within the integrase (int) and the attachment site (att) genes, both of which are essential for HIV-1 integration. Compared to shRNA targeting of the HIV-1 transcription factor tat (shTat), shRNA against int (shIN) or the U3 region of att (shU3) showed a more potent inhibitory effect on HIV-1 replication in human CD4+ T cells. Infection with a high dose of HIV-1 resulted in the emergence of escape mutants during long-term culture. Of note, limited genetic variation was observed in the viruses resistant to shIN. A combination of shINs against wild-type and escape mutant sequences had a negative effect on their antiviral activities, indicating a potentially detrimental effect when administering multiple shRNA targeting the same region to combat HIV-1 variants. The combination of shIN and shU3 att exhibited the strongest anti-HIV-1 activity, as seen by complete abrogation of viral DNA synthesis and viral integration. In addition, a modified long-hairpin RNA spanning the 50 nucleotides in the shIN target region effectively suppressed wild-type and shIN-resistant mutant HIV-1. These results suggest that targeting of incoming viral RNA before proviral DNA formation occurs through the use of nonoverlapping multiple siRNAs is a potent approach to achieving sustained, efficient suppression of highly mutable viruses, such as HIV-1.

scholarly journals Targeted Knockout of the Rickettsia rickettsii OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Nicholas F. Noriea ◽  
Tina R. Clark ◽  
Ted Hackstadt
Keyword(s):  
Guinea Pig ◽  
Stop Codon ◽  
Spotted Fever ◽  
Premature Stop Codon ◽  
Group Ii Intron ◽  
Pig Model ◽  
Spotted Fever Group ◽  
Wild Type ◽  
Group Ii ◽  
Guinea Pig Model

ABSTRACTStrains ofRickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), differ dramatically in virulence despite >99% genetic homology. Spotted fever group (SFG) rickettsiae produce two immunodominant outer membrane proteins, rickettsial OmpA (rOmpA) and rOmpB, which are conserved throughout the SFG and thought to be fundamental to pathogenesis. rOmpA is present in all virulent strains ofR. rickettsiibut is not produced in the only documented avirulent strain, Iowa, due to a premature stop codon. Here we report the creation of an isogenicompAmutant in the highly virulent strain Sheila Smith by insertion of intronic RNA to create a premature stop codon 312 bp downstream of the 6,747-bp open reading frame initiation site (int312). Targeted insertion was accomplished using an LtrA group II intron retrohoming system. Growth and entry rates of Sheila SmithompA::int312 in Vero cells remained comparable to those of the wild type. Virulence was assessed in a guinea pig model by challenge with 100 PFU of eitherompA::int312 Sheila Smith or the wild type, but no significant difference in either fever peak (40.5°C) or duration (8 days) were shown between the wild type and the knockout. The ability to disrupt genes in a site-specific manner using an LtrA group II intron system provides an important new tool for evaluation of potential virulence determinants in rickettsial disease research.IMPORTANCER. rickettsiirOmpA is an immunodominant outer membrane autotransporter conserved in the spotted fever group. Previous studies and genomic comparisons suggest that rOmpA is involved in adhesion and may be critical for virulence. Little information is available for rickettsial virulence factors in an isogenic background, as limited systems for targeted gene disruption are currently available. Here we describe the creation of an rOmpA knockout by insertion of a premature stop codon into the 5′ end of the open reading frame using a group II intron system. An isogenic rOmpA knockout mutation in the highly virulent Sheila Smith strain did not cause attenuation in a guinea pig model of infection, and no altered phenotype was observed in cell culture. We conclude that rOmpA is not critical for virulence in a guinea pig model but may play a role in survival or transmission from the tick vector.

scholarly journals Deletion of Rv2571c Confers Resistance to Arylamide Compounds in Mycobacterium tuberculosis

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Catherine D. Shelton ◽  
Matthew B. McNeil ◽  
Julie V. Early ◽  
Thomas R. Ioerger ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Stop Codon ◽  
Fusaric Acid ◽  
Premature Stop Codon ◽  
Acid Resistance ◽  
New Drugs ◽  
Wild Type ◽  
Content Type ◽  
Global Health Problem

ABSTRACT Tuberculosis, caused by Mycobacterium tuberculosis, is an urgent global health problem requiring new drugs, new drug targets, and an increased understanding of antibiotic resistance. We have determined the mode of resistance to be a series of arylamide compounds in M. tuberculosis. We isolated M. tuberculosis resistant mutants to two arylamide compounds which are inhibitory to growth under host-relevant conditions (butyrate as a sole carbon source). Thirteen mutants were characterized, and all had mutations in Rv2571c; mutations included a premature stop codon and frameshifts as well as nonsynonymous polymorphisms. We isolated a further 10 strains with mutations in Rv2571c with resistance. Complementation with a wild-type copy of Rv2571c restored arylamide sensitivity. Overexpression of Rv2571c was toxic in both wild-type and mutant backgrounds. We constructed M. tuberculosis strains with an unmarked deletion of the entire Rv2571c gene by homologous recombination and confirmed that these were resistant to the arylamide series. Rv2571c is a member of the aromatic amino acid transport family and has a fusaric acid resistance domain which is associated with compound transport. Since loss or inactivation of Rv2571c leads to resistance, we propose that Rv2571c is involved in the import of arylamide compounds.

Consequences of the Development of Nisin-Resistant Listeria monocytogenes in Fermented Dairy Products

2005 ◽  
Vol 68 (11) ◽  
pp. 2383-2388 ◽  
Author(s):  
BEATRIZ MARTÍNEZ ◽  
DIEGO BRAVO ◽  
ANA RODRÍGUEZ
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Cross Resistance ◽  
Clear Correlation ◽  
Wild Type ◽  
Northern Spain ◽  
Ph Values ◽  
Fermented Dairy Products ◽  
Fermented Dairy

Wild Listeria isolates representing serovars found in artisanal cheeses commercialized in Asturias (northern Spain) were assessed for their susceptibility to several bacteriocins. Pediocin PA-1 was the most active bacteriocin followed by enterocin AS-48, nisin, and plantaricin C. However, some Listeria monocytogenes and Listeria innocua strains were already highly resistant to PA-1. Among the wild L. monocytogenes populations, the frequency of development of nisin resistance ranged from 10−6 up to 10−3, depending on the strain. Highly stable mutants with increased nisin resistance (two- to fourfold) were isolated and tested for potential cross-resistance to lysozyme, EDTA, and various NaCl concentrations and pH values. All mutants were cross-resistant to lysozyme but sensitive to EDTA. In contrast, no clear correlation could be established between nisin resistance and an altered susceptibility to NaCl or pH changes. Nisin-resistant variants were able to survive and even to multiply in milk fermented by a nisin-producing Lactococcus, but the growth of the wild-type strain was inhibited. The different phenotypes evaluated in this study are indicative of the unpredictability of the consequences of the development of nisin resistance in a dairy environment. This resistance should be considered when making a risk assessment of the long-term use of nisin to control L. monocytogenes.

scholarly journals Novel Central Nervous System (CNS)-Targeting Protease Inhibitors for Drug-Resistant HIV Infection and HIV-Associated CNS Complications

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Masayuki Amano ◽  
Pedro Miguel Salcedo-Gómez ◽  
Ravikiran S. Yedidi ◽  
Rui Zhao ◽  
Hironori Hayashi ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Protease Inhibitors ◽  
Multidrug Resistant ◽  
Strong Hydrogen Bond ◽  
Term Therapy ◽  
Wild Type ◽  
Hiv 1 ◽  
Cns Targeting

ABSTRACT There is currently no specific therapeutics for the HIV-1-related central nervous system (CNS) complications. Here we report that three newly designed CNS-targeting HIV-1 protease inhibitors (PIs), GRL-083-13, GRL-084-13, and GRL-087-13, which contain a P1-3,5-bis-fluorophenyl or P1-para-monofluorophenyl ring, and P2-bis-tetrahydrofuran (bis-THF) or P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), with a sulfonamide isostere, are highly active against wild-type HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0002 to ∼0.003 μM), with minimal cytotoxicity. These CNS-targeting PIs efficiently suppressed the replication of HIV-1 variants (EC50, 0.002 to ∼0.047 μM) that had been selected to propagate at high concentrations of conventional HIV-1 PIs. Such CNS-targeting PIs maintained their antiviral activity against HIV-2ROD as well as multidrug-resistant clinical HIV-1 variants isolated from AIDS patients who no longer responded to existing antiviral regimens after long-term therapy. Long-term drug selection experiments revealed that the emergence of resistant-HIV-1 against these CNS-targeting PIs was substantially delayed. In addition, the CNS-targeting PIs showed the most favorable CNS penetration properties among the tested compounds, including various FDA-approved anti-HIV-1 drugs, as assessed with the in vitro blood-brain barrier reconstruction system. Crystallographic analysis demonstrated that the bicyclic rings at the P2 moiety of the CNS-targeting PIs form strong hydrogen-bond interactions with HIV-1 protease (PR) active site. Moreover, both the P1-3,5-bis-fluorophenyl and P1-para-monofluorophenyl rings sustain greater van der Waals contacts with PR than in the case of darunavir (DRV). The data suggest that the present CNS-targeting PIs have desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 strains and might serve as promising preventive and/or therapeutic candidates for HIV-1-associated neurocognitive disorders (HAND) and other CNS complications.

A new ferrochelatase mutation combined with low expression alleles in a Japanese patient with erythropoietic protoporphyria

2002 ◽  
Vol 102 (5) ◽  
pp. 501-506 ◽  
Author(s):  
Yumiko YASUI ◽  
Shikibu MURANAKA ◽  
Tsuyoshi TAHARA ◽  
Ryo SHIMIZU ◽  
Sonoko WATANABE ◽  
...  
Keyword(s):  
Base Pair ◽  
Japanese Patient ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Position ◽  
Molecular Defect ◽  
Wild Type ◽  
Erythropoietic Protoporphyria ◽  
Base Pair Deletion ◽  
Low Expression

We investigated the molecular defect of the ferrochelatase gene in a Japanese patient with erythropoietic protoporphyria (EPP), and identified a novel 16 base pair (574-589) deletion within exon 5. This deletion resulted in a frame-shift mutation and created a premature stop codon at amino acid position 198. The same molecular defect was also identified in his mother and a brother who had symptomatic EPP, but not in his father who was asymptomatic. The subjects with EPP were homozygous for the low expression haplotype, while his father was heterozygous for this haplotype. These results indicate that the combination of a 16 base pair deletion and low expression of the wild-type allelic variant is responsible for EPP in this pedigree.

scholarly journals The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

2013 ◽  
Vol 80 (2) ◽  
pp. 751-756 ◽  
Author(s):  
María Claudia Abeijón Mukdsi ◽  
Hélène Falentin ◽  
Marie-Bernadette Maillard ◽  
Victoria Chuat ◽  
Roxana Beatriz Medina ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Type Strain ◽  
Wild Type Strain ◽  
Lipolytic Activity ◽  
Premature Stop Codon ◽  
Propionibacterium Freudenreichii ◽  
Wild Type ◽  
Swiss Cheese ◽  
Content Type ◽  
Type Strains

ABSTRACTFree fatty acids are important flavor compounds in cheese.Propionibacterium freudenreichiiis the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis byP. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants ofP. freudenreichiiCIRM-BIA1Tfor each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of thepf279gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of thepf774gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of thepf279gene sequence, leading to a premature stop codon, whereas they harbored apf774gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase inP. freudenreichiiand a key agent of Swiss cheese lipolysis.

scholarly journals Pink Bollworm Resistance to Bt Toxin Cry1Ac Associated with an Insertion in Cadherin Exon 20

Toxins ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 186 ◽  
Author(s):  
Ling Wang ◽  
Yuemin Ma ◽  
Xueqin Guo ◽  
Peng Wan ◽  
Kaiyu Liu ◽  
...  
Keyword(s):  
Insect Pests ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Pink Bollworm ◽  
Cross Resistance ◽  
Bt Cotton ◽  
Wild Type ◽  
Bt Toxin ◽  
Exon 20 ◽  
In Cells

Insecticidal proteins from Bacillus thuringiensis (Bt) are widely used to control insect pests, but their efficacy is reduced when pests evolve resistance. We report on a novel allele (r16) of the cadherin gene (PgCad1) in pink bollworm (Pectinophora gossypiella) associated with resistance to Bt toxin Cry1Ac, which is produced by transgenic cotton. The r16 allele isolated from a field population in China has 1545 base pairs of a degenerate transposon inserted in exon 20 of PgCad1, which generates a mis-spliced transcript containing a premature stop codon. A strain homozygous for r16 had 300-fold resistance to Cry1Ac, 2.6-fold cross-resistance to Cry2Ab, and completed its life cycle on transgenic Bt cotton producing Cry1Ac. Inheritance of Cry1Ac resistance was recessive and tightly linked with r16. Compared with transfected insect cells expressing wild-type PgCad1, cells expressing r16 were less susceptible to Cry1Ac. Recombinant cadherin protein was transported to the cell membrane in cells transfected with the wild-type PgCad1 allele, but not in cells transfected with r16. Cadherin occurred on brush border membrane vesicles (BBMVs) in the midgut of susceptible larvae, but not resistant larvae. These results imply that the r16 allele mediates Cry1Ac resistance in pink bollworm by interfering with the localization of cadherin.

Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 907-915 ◽  
Author(s):  
Frank F. Weichold ◽  
Davide Zella ◽  
Oxana Barabitskaja ◽  
Jaroslaw P. Maciejewski ◽  
Daniel E. Dunn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Human Immunodeficiency Virus ◽  
Hematopoietic Stem Cells ◽  
Viral Gene ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Immunodeficiency Virus ◽  
Hiv 1

Abstract Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

A Leu262Pro mutation in the integrin β3 subunit results in an αIIb-β3 complex that binds fibrin but not fibrinogen

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 161-169 ◽  
Author(s):  
Christopher M. Ward ◽  
Anita S. Kestin ◽  
Peter J. Newman
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Surface Expression ◽  
Fibrin Clot ◽  
Wild Type ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
293 Cells ◽  
Platelet Disorder

Abstract Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, IIbβ3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant IIbβ3 may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable IIbβ3, findings consistent with type II GT. Genotyping of LD revealed 2 novel β3 mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among β integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Proβ3 cotransfected with wild-type IIb into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Proβ3 formed a complex with endogenous av and retracted fibrin clots similarly to wild-type β3. The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for IIbβ3 to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding β3 Leu262 may maintain β3 in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.

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