INVESTIGATION OF SPECIFIC AND NON-SPECIFIC RNA SECONDARY STRUCTURE UNFOLDING ACTIVITY OF HRSV M2-1 PROTEIN AND ITS GLOBULAR DOMAIN

The human Respiratory Syncytial Virus is one of the main causative agents of acute respiratory diseases, such as bronchiolitis and pneumonia in children worldwide. In the viral transcription process, hRSV M2-1 protein acts as a processivity and antitermination factor, preventing the RNA polymerase complex from dissociating upon reaching the stop codon and, therefore, increasing the transcription efficiency of genes near the 5' end. Evidence suggests that M2-1 acts by binding to nascent mRNA, facilitating the elongation of the transcript, preventing it from rehybridizing with the template strand, or forming secondary structures that could destabilize the transcription complex. This hypothesis is supported by studies reporting the unfolding/destabilization activity of short and long RNA secondary structures promoted by M2-1 and its core domain (cdM2-1), respectively. Despite these initial findings, it is worth noting that M2-1/dgM2-1 activity is not completely understood. The present study aimed to characterize the RNA secondary structure unfolding/destabilization activity of M2-1 and dgM2-1. Initially, UV-Vis absorption experiments were performed to characterize the interaction of sequence-specific (double-stranded polyA-polyU) and non-specific (yeast RNA) RNA with the DAPI probe, a molecule that binds exclusively to regions of the secondary structure of nucleic acids. The interaction of both RNAs with DAPI was followed by a hypochromic and red-shift effect of the spectral band at 343 nm of the probe. Next, DAPI/RNA complexes were placed in the presence of M2-1 and dgM2-1 to investigate the unfolding activity of the RNAs using DAPI as a probe. The presence of M2-1 and dgM2-1 in the DAPI/RNA complex solution promoted a blue-shift effect of the absorption band towards the previous value of 343 nm of free probe, indicating that M2-1 and dgM2-1 present RNA unfolding activity. The most significant activity was observed for M2-1 in comparison to dgM2-1, being M2-1 activity was more efficient for the non-specific sequence RNA concerning the specific. Therefore, the results of the present work contribute to a better understanding of the key role of M2-1 in the context of the viral co-transcriptional complex and, consequently, in the replication process of hRSV. 

This work was supported by FAPESP (2022/13050-5, 2023/06367-5, 2023/09642-7, and 2023/18111-5), CNPq (317157/2023-0), and FINEP