Study of competitive interaction between steroidal alkaloids and RNA for the transcription anti-termination factor binding site of human respiratory syncytial virus (hRSV)

Abstract

Human respiratory syncytial virus (hRSV) is the main agent present in lower respiratory tract infections in children and elderly people worldwide. Currently, the pathologies caused by RSV are poorly understood and vaccine development data are scarce. The virus has a negative single stranded, non segmented RNA genome of 15. 2 kb and ten genes, which encode eleven proteins, due to two overlapping open reading frames located in the M2 gene, producing the M2-1 and M2- 2 proteins. The M2-1 protein is essential for hRSV viral cycle maintenance and acts as a factor of processivity and anti-termination, stabilizing and preventing viral RNA polymerase from dissociating early from the RNA, thus increasing both the efficiency of replication as well as transcription, especially of genes near the 5' end. A published study showed that steroidal alkaloids, such as cyclopamine, had significant antiviral activity both in vitro and in vivo. The same group observed that the alkaloid affected the viral RNA polymerase complex efficiency and attributed this phenomenon to the lower expression of the transcriptional anti-termination factor, the M2-1 protein, but the authors did not describe the protein/ligand interaction mechanism. Performing site-directed mutation experiments on this protein, it was discovered that the virus has become resistant to treatments with the alkaloid. Thus, there is a possibility that other compounds structurally similar to cyclopamine, like solasodine, play similar roles. Therefore, the M2-1 protein probably has a site for the binding of a steroidal alkaloid, such as cyclopamine and solasodine. This site coincides with an important region for the interaction between the protein and RNA in the globular domain of the protein (dM2-1), hence the alkaloid is an antiviral candidate against hRSV infection. In parallel, our group succeeded in expressing recombinant hRSV M2-1 and dM2-1 proteins. The main objective of the project is to elucidate the mechanism of interaction between these alkaloids and the protein at the RNA binding site. This study will be performed by fluorescence spectroscopy and nuclear magnetic resonance, techniques that will allow to calculate the protein/ligand binding constants, to characterize the interaction type and epitopes and thus obtain information about the molecular mechanism of the interaction. The results obtained in our study, together with the results found in the literature about in vivo and in vitro experiments, will allow to propose efficient models of interaction and therapeutics. The present proposal is alligned with the epidemiological scenario that claims for the maintenance of scientific efforts in the search for efficient anti-RSV drugs. (AU)