DEVELOPMENT OF AN ALBUMIN-BINDING DOMAIN CARRIER AND A NOVEL PEPTIDE MIMETIC ANTIVIRAL FOR RESPIRATORY SYNCYTIAL VIRUS
Notice bibliographique
Résumé
Background: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection and hospitalization in children worldwide. With no vaccine or antivirals available for the routine prevention or treatment of RSV, an effective RSV antiviral is required. Previous studies have shown that the RSV nucleocapsid complex (NC), phosphoprotein (P), and large polymerase (L) are essential for the replication and survival of RSV since they form the core of the RNA-dependent RNA polymerase (RdRp) complex. Thus, these proteins are viable targets for novel RSV antivirals. Objective: The Mahony laboratory has previously shown that 20 µM of a peptide mimetic composed of the 21 terminal amino acids of the RSV phosphoprotein (RSVP220-241) fused to an HIV-1 Tat cell penetrating peptide (CPP), a hexa-histidine (His) tag, and the Escherichia coli (E. coli) maltose binding protein carrier (MBP) molecule was sufficient to attenuate RSV A and B replication in vitro by approximately 90 and 80%, respectively. We evaluated the fusion of this His-MBP-Tat-RSVP220-241 mimetic to a more suitable carrier molecule, an albumin-binding domain (ABD), for future use in vivo. In addition, we designed a novel antiviral mimetic composed of the 30 terminal amino acids of the RSV A P protein (RSVP212-241), which are involved in binding both L polymerase and NC complexes, fused to a CPP consisting of Tat or nine arginine residues (Arg9), a His-tag, and the MBP carrier. We evaluated the activity of His-MBP-Tat-RSVP212-241, Tat-His-MBP-Tat-RSVP212-241, and His-Arg9-MBP-RSVP212-241 mimetics in vitro and hypothesized that a mimetic designed to target both L and NC interactions would be a more effective RSV antiviral than the original His-MBP-Tat-RSVP220-241 mimetic. Methods and Results: The Gateway® Cloning System was used to create expression vectors containing His-, GST-, or His-MBP-ABD-Tat-RSVP220-241 and His-MBP-Tat-RSVP212-241, whereas inverse PCR and both the In-Fusion® and Gateway® Cloning systems were used to generate expression vectors containing Tat-His-MBP-Tat-RSVP212-241 and His-Arg9-MBP-RSVP212-241. The fusion proteins were expressed, purified by affinity chromatography, and evaluated in vitro. No soluble protein was obtained for the ABD constructs. His-MBP-Tat-RSVP212-241 was toxic and not internalized by LLC-MK2 cells, whereas only 0.26 mg of Tat-His-MBP-Tat-RSVP212-241 was purified. We were able to show that His-Arg9-MBP-RSVP212-241 was non-toxic, internalized, and interacted with the RSV nucleoprotein (N) in a GST pull-down experiment. Furthermore, His-Arg9-MBP-RSVP212-241 attenuated RSV A replication and progeny production by 94.8 and 93.33% at 200 µM, respectively. We demonstrated 50.7 and 49% inhibition of RSV A replication and progeny production at 20 µM, respectively. We showed that inhibition of viral replication by 25 µM His-Arg9-MBP-RSVP212-241 was not significantly different from inhibition by 20 µM His-MBP-Tat-RSVP220-241. Thus, in this thesis we were unable to show that His-Arg9-MBP-RSVP212-241 was a more effective RSV antiviral. Conclusion: The ABD was not a suitable carrier molecule for use with our fusion protein mimetics. However, RSV P protein mimetics that target interactions with the NC complexes and L polymerase are a novel and viable antiviral strategy. We showed that a His-Arg9-MBP-RSVP212-241 mimetic was non-toxic, internalized, and interacted with the RSV N protein in vitro. Furthermore, we showed that at 200 µM this novel mimetic could attenuate RSV A replication and progeny production in vitro by 94.8 and 93.3%, respectively. Further studies are required to characterize the construct, increase its bioactivity, and identify a suitable human carrier molecule for future evaluation in vivo.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».