scholarly journals SilentMutations (SIM): a tool for analyzing long-range RNA-RNA interactions in viral genomes and structured RNAs

2018 ◽  
Author(s):  
Daniel Desirò ◽  
Martin Hölzer ◽  
Bashar Ibrahim ◽  
Manja Marz
Keyword(s):  
Long Range ◽  
Influenza A ◽  
Graphical Representation ◽  
Point Mutations ◽  
Multiple Point ◽  
Coding Region ◽  
Viral Rnas ◽  
Viral Genomes ◽  
Single Nucleotide Change

ABSTRACTBackgroundA single nucleotide change in the coding region can alter the amino acid sequence of a protein. In consequence, natural or artificial sequence changes in viral RNAs may have various effects not only on protein stability, function and structure but also on viral replication.In recent decades, several tools have been developed to predict the effect of mutations in structured RNAs such as viral genomes or non-coding RNAs. Some tools use multiple point mutations and also take coding regions into account. However, none of these tools was designed to specifically simulate the effect of mutations on viral long-range interactions.ResultsHere, we developedSilentMutations (SIM), an easy-to-use tool to analyze the effect of multiple point mutations on the secondary structures of two interacting viral RNAs. The tool can simulate disruptive and compensatory mutants of two interacting single-stranded RNAs. This allows a fast and accurate assessment of key regions potentially involved in functional long-range RNA-RNA interactions and will eventually help virologists and RNA-experts to design appropriate experiments.SIMonly requires two interacting single-stranded RNA regions as input. The output is a plain text file containing the most promising mutants and a graphical representation of all interactions.ConclusionWe applied our tool on two experimentally validated influenza A virus and hepatitis C virus interactions and we were able to predict potential double mutants forin vitrovalidation experiments.AvailabilityThe source code and documentation ofSIMare freely available at github.com/desiro/silentMutations.

scholarly journals Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

2014 ◽  
Vol 95 (5) ◽  
pp. 1033-1042 ◽  
Author(s):  
Blanca García-Barreno ◽  
Teresa Delgado ◽  
Sonia Benito ◽  
Inmaculada Casas ◽  
Francisco Pozo ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A ◽  
Point Mutations ◽  
Antigenic Site ◽  
Single Amino Acid ◽  
Antigenic Structure ◽  
Human Antibody ◽  
2009 H1n1

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283–6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

scholarly journals Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro

2007 ◽  
Vol 82 (2) ◽  
pp. 1059-1063 ◽  
Author(s):  
Wai-Hong Wu ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Coding Region ◽  
Epitope Tag ◽  
Carboxy Terminal

ABSTRACT A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

scholarly journals Variants in the 5′UTR reduce SHOX expression and contribute to SHOX haploinsufficiency

2020 ◽  
Vol 29 (1) ◽  
pp. 110-121
Author(s):  
Deepak Babu ◽  
Silvia Vannelli ◽  
Antonella Fanelli ◽  
Simona Mellone ◽  
Ave Maria Baffico ◽  
...  
Keyword(s):  
Point Mutations ◽  
Mrna Splicing ◽  
Luciferase Assay ◽  
Transcriptional Level ◽  
Coding Region ◽  
Exon Trapping ◽  
Branch Site ◽  
Uncertain Significance ◽  
Negative Effect

AbstractSHOX haploinsufficiency causes 70–90% of Léri-Weill dyschondrosteosis (LWD) and 2–10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5′UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5′UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5′UTR.

scholarly journals Cell Membrane Adaptations Mediate β-Lactam-Induced Resensitization of Daptomycin-Resistant (DAP-R) Staphylococcus aureus In Vitro

2021 ◽  
Vol 9 (5) ◽  
pp. 1028
Author(s):  
Nagendra N. Mishra ◽  
Arnold S. Bayer ◽  
Sarah L. Baines ◽  
Ashleigh S. Hayes ◽  
Benjamin P. Howden ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cell Membrane ◽  
Surface Charge ◽  
Prolonged Exposure ◽  
Point Mutations ◽  
Carotenoid Content ◽  
Multiple Point ◽  
Genetic Changes ◽  
Allelic Exchange

The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected β-lactams occurs coincident with the accumulation of multiple point mutations in the mprF gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such β-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin (‘daptomycin-resensitized’). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected β-lactams results in both definable genetic changes (i.e., mprF mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.

scholarly journals Structures and Functions of Viral 5’ Non-Coding Genomic RNA Domain-I in Enterovirus-B Infections

2020 ◽  
Author(s):  
Marie GLENET ◽  
Laetitia HENG ◽  
Domitille CALLON ◽  
Anne-Laure LEBREIL ◽  
Paul-Antoine GRETTEAU ◽  
...  
Keyword(s):  
Viral Replication ◽  
Secondary Structures ◽  
Genomic Rna ◽  
Coding Region ◽  
Viral Genomes ◽  
Rna Domains ◽  
Group B ◽  
Human Infections ◽  
Domain I

Group-B enteroviruses (EV-B) are ubiquitous naked single-stranded positive RNA viral pathogens that are responsible for common acute or persistent human infections. Their genome is composed in the 5'end by a non-coding region, which is crucial for the initiation of the viral replication and translation processes. RNA domain-I secondary structures can interact with viral or cellular proteins to form viral ribonucleoprotein (RNP) complexes regulating viral genomic replication, whereas RNA domains-II to -VII (IRES) are known to interact with cellular ribosomal subunits to initiate the viral translation process. Natural 5&rsquo; terminally deleted viral forms lacking some genomic RNA domain-I secondary structures have been described in EV-B induced murine or human infections. Recent in vitro studies have evidenced that the loss of some viral RNP complexes in the RNA domain-I can modulate the viral replication and infectivity levels in EV-B infections. Moreover, the disruption of secondary structures of RNA domain-I could impair viral RNA sensing by RIG-I or MDA5 receptors, a way to overcome antiviral innate immune response. Overall, natural 5&prime; terminally deleted viral genomes resulting in the loss of various structures in the RNA domain-I could be major key players of host-cell interactions driving the development of acute or persistent EV-B infections.

scholarly journals Molecular cloning and characterization of human caspase-activated DNase

1998 ◽  
Vol 95 (16) ◽  
pp. 9123-9128 ◽  
Author(s):  
Naomi Mukae ◽  
Masato Enari ◽  
Hideki Sakahira ◽  
Yoji Fukuda ◽  
Johji Inazawa ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Cell Lines ◽  
Point Mutations ◽  
Leukemic Cell ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Caspase Activated Dnase

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.

scholarly journals Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication

2021 ◽  
Vol 118 (13) ◽  
pp. e2020969118
Author(s):  
Cyril Le Nouën ◽  
Thomas McCarty ◽  
Lijuan Yang ◽  
Michael Brown ◽  
Eckard Wimmer ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
De Novo ◽  
Point Mutations ◽  
Surface Glycoprotein ◽  
Temperature Sensitive ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Codon Pair ◽  
Syncytial Virus

Recoding viral genomes by introducing numerous synonymous but suboptimal codon pairs—called codon-pair deoptimization (CPD)—provides new types of live-attenuated vaccine candidates. The large number of nucleotide changes resulting from CPD should provide genetic stability to the attenuating phenotype, but this has not been rigorously tested. Human respiratory syncytial virus in which the G and F surface glycoprotein ORFs were CPD (called Min B) was temperature-sensitive and highly restricted in vitro. When subjected to selective pressure by serial passage at increasing temperatures, Min B substantially regained expression of F and replication fitness. Whole-genome deep sequencing showed many point mutations scattered across the genome, including one combination of six linked point mutations. However, their reintroduction into Min B provided minimal rescue. Further analysis revealed viral genomes bearing very large internal deletions (LD genomes) that accumulated after only a few passages. The deletions relocated the CPD F gene to the first or second promoter-proximal gene position. LD genomes amplified de novo in Min B–infected cells were encapsidated, expressed high levels of F, and complemented Min B replicationin trans. This study provides insight on a variation of the adaptability of a debilitated negative-strand RNA virus, namely the generation of defective minihelper viruses to overcome its restriction. This is in contrast to the common “defective interfering particles” that interfere with the replication of the virus from which they originated. To our knowledge, defective genomes that promote rather than inhibit replication have not been reported before in RNA viruses.

scholarly journals A Hairpin Loop at the 5′ End of Influenza A Virus Virion RNA Is Required for Synthesis of Poly(A)+ mRNA In Vitro

1999 ◽  
Vol 73 (3) ◽  
pp. 2109-2114 ◽  
Author(s):  
David C. Pritlove ◽  
Leo L. M. Poon ◽  
Louise J. Devenish ◽  
Mike B. Leahy ◽  
George G. Brownlee
Keyword(s):  
Nucleotide Sequence ◽  
Influenza A Virus ◽  
Influenza A ◽  
Point Mutations ◽  
Evidence Based ◽  
Hairpin Loop ◽  
Base Pairs ◽  
Present Evidence ◽  
Polyadenylated Mrna

ABSTRACT We present evidence, based on extensive mutagenesis, that a hairpin loop at the 5′ end of influenza A virus virion RNA (vRNA) is required for the synthesis of polyadenylated mRNA from model vRNA templates in vitro. The hairpin loop, which we term the vRNA 5′ hook, contains a stem of 2 bp formed by the second and third residues pairing with the ninth and eighth residues, respectively, and a 4-nucleotide loop composed of the intervening residues 4 to 7. Disruption of the base pairs of the vRNA 5′ hook by introducing point mutations prevented polyadenylation, except in two mutants where a G-U base pair reformed. The polyadenylation activity of point mutants could be rescued by constructing double mutants with reformed base pairs in the stem of the vRNA 5′ hook. These results suggest that base pairing rather than a particular nucleotide sequence was critical. We also show that mutation of the analogous region in the 3′ arm of vRNA did not interfere with the synthesis of polyadenylated mRNA, suggesting that a hook structure in the 3′ arm is not required for transcription of polyadenylated mRNA in vitro.

scholarly journals Temperature-Sensitive Lesions in Two Influenza A Viruses Defective for Replicative Transcription Disrupt RNA Binding by the Nucleoprotein

1999 ◽  
Vol 73 (9) ◽  
pp. 7349-7356 ◽  
Author(s):  
Liz Medcalf ◽  
Emma Poole ◽  
Debra Elton ◽  
Paul Digard
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Point Mutations ◽  
Temperature Shift ◽  
Binding Activity ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Coding Region ◽  
Influenza A Viruses

ABSTRACT The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30°C. This suggests that the RNA-binding activity of NP is required for replicative transcription.

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