The human Respiratory Syncytial Virus (hRSV) is the most frequent agent of respiratory infections in infants and children with no currently approved vaccine. The M_2-1 protein is an important transcriptional antitermination factor and a potential target for viral replication inhibitor development. Hesperetin (HST) and hesperidin (HSD) are flavonoids from the flavanone group, naturally found in citrus and have, as one of their properties, antiviral activity. The present study reports on the interactions between hRSV M_2-1 and these flavanones using experimental techniques in association with computational tools. STD-NMR results showed that HST and HSD bind to M_2-1 by positioning their aromatic rings into the target protein binding site. Fluorescence quenching measurements revealed that HST had an interaction affinity greater than HSD towards M_2-1. The thermodynamic analysis suggested that hydrogen bonds and van der Waals interactions are important for the molecular stabilization of the complexes. Computational simulations corroborated with the experimental results and indicated that the possible interaction region for the flavonoids is the AMP-binding site in M_2-1. Therefore, these results point that HST and HSD bind stably to a critical region in M_2-1, which is vital for its biological function, and thus might play a possible role antiviral against hRSV.

The human Respiratory Syncytial Virus (hRSV) stands as one of the most common causes of acute respiratory diseases. The infectivity of this virus is intricately linked to its membrane proteins, notably the attachment glycoprotein (G protein). The latter plays a key role in facilitating the attachment of hRSV to respiratory tract epithelial cells, thereby initiating the infection process. The present study aimed to characterize the interaction of the conserved cysteine-noose domain of hRSV G protein (cndG) with the transmembrane CX3C motif chemokine receptor 1 (CX3CR1) isoforms using computational tools of molecular modeling, docking, molecular dynamics simulations, and binding free energy calculations. From MD simulations of the molecular system embedded in the POPC lipid bilayer, we showed a stable interaction of cndG with the canonical fractalkine binding site in the N-terminal cavity of the CX3CR1 isoforms and identified that residues in the extracellular loop 2 (ECL2) region and Glu279 of this receptor are pivotal for the stabilization of CX3CR1/cndG binding, corroborating what was reported for the interaction of the chemokine fractalkine with CX3CR1 and its structure homolog US28. Therefore, the results presented here contribute by revealing key structural points for the CX3CR1/G interaction, allowing us to better understand the biology of hRSV from its attachment process and to develop new strategies to combat it.

The respiratory syncytial virus (RSV) matrix protein plays key roles in the virus life cycle and is essential for budding, as it stimulates the optimal membrane curvature necessary for the emergence of viral particles. Resveratrol, a polyphenol (3,4′,5-trihydroxy-trans-stilbene) produced by plants, exhibits pharmacological effects, including anti-inflammatory and antiviral activities. In this study, resveratrol was tested in HEp-2 (Epidermoid carcinoma of the larynx cell) cells for its post-infection effects, and recombinant M protein was produced to characterize the biophysical mechanisms underlying this interaction. The CC50 (Cytotoxic concentration 50%) value for resveratrol was determined to be 297 μM over 48 h, and the results from the HEp-2 cell cultures demonstrated a viral inhibition of 42.7% in the presence of resveratrol, with an EC50 (Half maximal effective concentration) of 44.26 μM. This mechanism may occur through interaction with the M protein responsible for the budding of mature viral particles. Biophysical assays enabled us to characterize the interaction of the M/resveratrol complex as an entropically driven bond, guided by hydrophobic interactions at the dimerization interface of the M protein, which is essential for the stabilization and formation of the oligomers necessary for viral budding. These findings suggest that one of the targets for resveratrol binding is the M protein, indicating a potential site for blocking the progression of the infection.

Respiratory syncytial virus (RSV) is one of the most prevalent viruses that causes severe acute lower respiratory tract infections (ALRTIs) in the elderly and young children. There is no specific drug to treat RSV, only a broad-spectrum antiviral, ribavirin, which is only used in critical cases. Our research group is investigating antiviral agents of natural origin, such as coumarins and flavonoids, that may help reduce or prevent RSV infection. The cytotoxic concentrations of coumarins and hesperetin were tested on A549 and HEp-2 cells and used in inhibition tests in which 80% of the cells were viable. The anti-RSV action of the molecules was analyzed in A549 and HEp-2 cells and in HBE cell cultures infected with RSV-luc or rgRSV. We also encapsulated the compounds using β-cyclodextrin to improve the permeability and solubility of the molecules. Esculetin and 4-methyl inhibited rgRSV effectively on A549 and HEp-2 cells after 24 hpi, and when they were encapsulated, coumarin, esculetin, and hesperetin presented inhibition against rgRSV in HBE culture. The coumarins inhibit RSV replication in cell culture and even manage to overcome the mucus barriers of the HBE cultures, and β-cyclodextrin was essential for some of the coumarins to enter the cell and therefore to reach their targets.

Anais do XXXII Congresso Brasileiro de Virologia: Virologia em Casa (978-65-5941-393-5) - Use Of Resveratrol As Therapy Against Human Respiratory Syncytial Virus