An Efficient RNA Aptamer against Human Influenza B Virus Hemagglutinin

Gopinath, Subash C.B.; Sakamaki, Yuriko; Kawasaki, Kazunori; Kumar, Penmetcha K. R.

doi:10.1093/jb/mvj095

Cited by 108 publications

(71 citation statements)

References 24 publications

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“…The potential for small RNAs to act as protein agonists has long been appreciated, as demonstrated by the directed evolution of RNA aptamers that inhibit HIV reverse transcriptase (26) and influenza virus B hemagglutinin (27), among others (28). Despite the successful experimental generation of these artificial species, there are very few examples of naturally occurring RNAs that act directly to impede protein activity in vivo, and ToxI antitoxins are, to our knowledge, the only RNAs whose function is to inhibit their own parent-processing enzyme.…”

Section: Discussionmentioning

confidence: 99%

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot

Short

Pei

Blower

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial small RNAs perform numerous regulatory roles, including acting as antitoxic components in toxin-antitoxin systems. In type III toxin-antitoxin systems, small processed RNAs directly antagonize their toxin protein partners, and in the systems characterized the toxin and antitoxin components together form a trimeric assembly. In the present study, we sought to define how the RNA antitoxin, ToxI, inhibits its potentially lethal protein partner, ToxN. We show through cross-inhibition experiments with the ToxIN systems from Pectobacterium atrosepticum (ToxIN Pa ) and Bacillus thuringiensis (ToxIN Bt ) that ToxI RNAs are highly selective enzyme inhibitors. Both systems have an "addictive" plasmid maintenance phenotype. We demonstrate that ToxI Pa can inhibit ToxN Pa in vitro both in its processed form and as a repetitive precursor RNA, and this inhibition is linked to the self-assembly of the trimeric complex. Inhibition and self-assembly are both mediated entirely by the ToxI Pa RNA, with no requirement for cellular factors or exogenous energy. Finally, we explain the origins of ToxI antitoxin selectivity through our crystal structure of the ToxIN Bt complex. Our results show how a processed RNA pseudoknot can inhibit a deleterious protein with exquisite molecular specificity and how these self-contained and addictive RNA-protein pairs can confer different adaptive benefits in their bacterial hosts.RNA inhibition | mRNA interferase | RNA-protein complex | abortive infection | plasmid stabilization

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Section: Discussionmentioning

confidence: 99%

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot

Short

Pei

Blower

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…After the RNA was incubated with the target protein at room temperature for 10 min, the protein-RNA complexes were filtered through a prewetted nitrocellulose acetate filter (HAWP filter, 0.45 m and 13.0 mm; Millipore) fitted in a "pop-top" filter holder (Nuclepore) and washed with 1 ml binding buffer. The RNAs that were retained on the filter were eluted and recovered as previously described (14,15). The recovered RNAs were reverse transcribed in 20 l of a reaction mixture containing 50 mM Tris-HCl (pH 8.0), 40 mM KCl, 6 mM MgCl 2 , 0.4 mM deoxynucleoside triphosphates (dNTPs), 2.5 M primer, and 5 U avian myoblastosis virus (AMV) reverse transcriptase (Seikagaku).…”

Section: Viral Proteinsmentioning

confidence: 99%

“…32 P]ATP, and binding studies were performed by using a filter binding assay similar to that previously reported (15,21,22). The binding and in vitro transcription conditions were similar to those used for selection, except for the RNA and gD protein concentrations.…”

Section: Viral Proteinsmentioning

confidence: 99%

“…Previously, we selected aptamers that bound to the hemagglutinin (HA) of influenza A (H3N2) and B viruses with high specificity and affinity. These aptamers efficiently inhibited membrane fusion when challenged in vitro with whole viruses (14,15). Interestingly, the anti-influenza A virus aptamer displayed a high level of molecular discrimination of closely related HA proteins derived from other strains within the same subtype of influenza viruses (14).…”

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confidence: 99%

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Aptamer That Binds to the gD Protein of Herpes Simplex Virus 1 and Efficiently Inhibits Viral Entry

Gopinath

Hayashi

Kumar

2012

J Virol

103

107

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The ectodomain of the gD protein of herpes simplex viruses (HSVs) plays an important role in viral entry by binding to specific cellular coreceptors and mediating viral entry to the host cells. In the present study, we isolated RNA aptamers (aptamer-1 and aptamer-5) that specifically bind to the gD protein of HSV-1 with high affinity and are able to discriminate the gD protein of a different virus, HSV-2. Aptamer-1 efficiently interfered with the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner. The 50% effective concentration (EC 50 ) of aptamer-1 was estimated to be in the nanomolar range (60 nM). Furthermore, aptamer-1 was analyzed for anti-HSV-1 activity by using plaque assays, and it efficiently inhibited viral entry with an estimated K i of 0.8 M. To expand the future applications of aptamer-1, a shorter variant was designed by using both mapping and boundary analyses, resulting in the mini-1 aptamer (44-mer). Compared to the fulllength aptamer, mini-1 had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions. These studies suggest that the mini-1 aptamer could be explored further as an anti-HSV-1 topical therapy designed to prevent the risk of acquiring HSV-1 infection through physical contact. Herpes simplex virus 1 (HSV-1) and HSV-2 are widespread human pathogens that infect primarily epithelial tissues before spreading to the nervous system, where they become latent. The genomes of HSV-1 and HSV-2 share 50 to 70% homology, and the two viruses also share several cross-reactive epitopes. The entry of HSV into the host cell begins with the binding of viral proteins gC and gB to proteoglycans on the host cell surface. This binding interaction is then followed by the coordinated action of four essential glycoproteins: gD, gB, gH, and gL (3, 30). Of these four glycoproteins, the gD protein is required for binding to the specific cellular receptors for viral entry. The gD protein recognizes two protein receptors, herpesvirus entry mediator (HVEM) (34) and nectin-1 (25). In addition to these receptor proteins, HSV-1 entry can also be mediated by 3-O-sulfated-modified heparan sulfate (29). The gD proteins of HSV-1 and HSV-2 are highly homologous proteins, with a sequence identity of around 86% within the ectodomain region (amino acids [aa] 1 to 319).The structures of the gD protein of HSV-1 have been solved, both in the absence (Fig. 1a) (4) and in the presence (Fig. 1b) (20) of its cognate receptor, HVEM. Recently, the structure of gD bound to another receptor, nectin-1, was also determined by two groups (Fig. 1c and d) (8, 35). Those studies revealed that gD contains a V-like Ig fold between residues 56 and 184. This structural feature is commonly found in cell surface adhesion molecules. N-and C-terminal extensions flank this fold and are unfolded and disordered in the absence of the gD receptor. However, in the HVEM-bound form, the N terminus of gD folds into a hairpin structure that binds to HVEM (4). This recept...

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“…In another separate report, the same group has also utilized the same selection approach to generate RNA aptamers against the HA of human influenza B (a total of 9 selection cycles performed). [15] Among the obtained sequences, one of the aptamer was observed to demonstrate high selectivity as it is able to differentiate between the HA from influenza A and B viruses. Again via SPR, its K d was measured to be ~720 pM and, similar to human influenza A, the generated RNA aptamer is observed to be able to inhibit HA-mediated membrane fusion process for human influenza B.…”

Section: Bacillus Anthracis Spores (Cat-a Pathogen)mentioning

confidence: 99%

Aptasensors for biosecurity applications

Fischer

Tarasow

Tok

2007

Current Opinion in Chemical Biology

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SUMMARY:Nucleic acid aptamers have found steadily increased utility and application steadily over the last decade. In particular, aptamers have been touted as a valuable complement to and in some cases replacement for antibodies due to their structural and functional robustness as well as their ease in generation and synthesis. They are thus attractive for biosecurity applications, e.g. pathogen detection, and are especially well suited since their in vitro generation process does not require infection of any host systems. Herein we provide a brief overview of the aptamers generated against biopathogens over the last few years. In addition, a few recently described detection platforms using aptamers (aptasensors) and potentially suitable for biosecurity applications will be discussed.

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An Efficient RNA Aptamer against Human Influenza B Virus Hemagglutinin

Cited by 108 publications

References 24 publications

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot

Aptamer That Binds to the gD Protein of Herpes Simplex Virus 1 and Efficiently Inhibits Viral Entry

Aptasensors for biosecurity applications

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