Estruturas secundárias G-quadruplex no genoma do vírus Mayaro como alvo para estratégias antivirais

Abstract

The Mayaro virus (MAYV) is a mosquito-borne arthritogenic alphavirus and the etiologic agent of Mayaro fever. The infection is characterized by debilitating clinical manifestations, such as fever, myalgia, prolonged arthralgia, and ocular pain. MAYV circulates in several countries across the Americas and notably in Brazil, where cases are detected annually. Currently, there are no licensed antiviral drugs for the treatment of this viral infection. In this context, G-quadruplexes (G4s), secondary structures formed within viral genomes, have gained prominence due to their involvement in controlling essential processes of the viral replication cycle, emerging as potential therapeutic targets. Therefore, the present study aimed to identify G4 structures in the MAYV genome and evaluate their potential as targets for antiviral strategies. To achieve this goal, in silico analyses using the predictors QGRS Mapper, pqsfinder, and G4Hunter initially identified putative G4-forming sequences (PQS) within the genomes of genotypes L and D. Alignment of the complete genomic sequences revealed the presence of conserved PQS exclusive to genotypes L and D, as well as sequences common across all genotypes. Based on these predictions, the PQS located in the coding region of the nsP2 protein (RNA_3411) was selected for structural characterization. Circular dichroism (CD) analyses suggested that this sequence adopts a hybrid and antiparallel topological profile in the presence of stabilizing ions. Additionally, dose-response assays in Vero E6 cells infected with MAYV-NanoLuc demonstrated that treatment with the G4 ligands TMPyP4, BRACO-19, and NMM inhibited viral replication in a dose-dependent manner, resulting in selectivity indices (SI) of >12.4, 2.0, and >2.5, respectively. Furthermore, time-of-drug-addition (ToDA) assays revealed that TMPyP4 exhibits antiviral activity during the post-entry stage of the replication cycle, significantly reducing intra- and extracellular viral titers. Complementary CD assays showed that the interaction between TMPyP4 and RNA_3411 induces conformational rearrangements in the G4 topologies. Finally, assays using antisense oligonucleotides (ASO) targeting MAYV PQS inhibited viral replication by approximately 50% when used alone, and did not interfere with the activity of the TMPyP4 ligand when co-administered. Our results demonstrate the formation of G4 structures in the MAYV genome and suggest that the modulation of these structures by ligands represents a promising antiviral strategy.