Regulatory role of stem-loop structures in Faba bean necrotic yellows virus replication efficiency

Multipartite viruses package their genomic segments independently and primarily target plant hosts. A limited number of viruses are identified as targeting animals. The <i>Nanoviridae</i> is a family of multipartite single-stranded DNA (ssDNA) plant viruses that encapsidate approximately 1 kb ssDNA segments individually and transmit them via aphids in a circulative, non-propagative manner. Nanoviruses cause significant diseases in host plants, particularly in leguminous crops. Each segment of a nanovirus encodes an open reading frame that functions independently in the infection process. Additionally, each segment contains conserved inverted repeat sequences that create a stem-loop structure, along with a conserved nonanucleotide sequence known as "TAGTATTAC" located within the common region. This research analyzed the variations in the length of the stem-loop neck region of DNA-R segments of Faba bean necrotic yellows virus (FBNYV) and their implications using molecular dynamics (MD) simulations and experimental techniques. Although force field approximations and simulation time scales limit the quality of MD simulations, explicit solvent MD simulations successfully examined key characteristics of the stem-loop structure. The current study focused on the design of mutants based on variations in the neck region length of the stem-loop, construction of mutant infectious clones, and inoculation into the host plant, followed by expression patterns analysis of mutant stem-loop structures. The mutated stem-loop structures showed greater conformational stability compared to the wild type, and these variations in stability correspond to differences in FBNYV expression in the host plant. This finding establishes a framework for further structural and functional analyses of stem-loop structures of nanovirus.IMPORTANCEOur stem-loop modification experiments in Faba bean necrotic yellows virus (FBNYV) highlight its critical role in viral replication and genome stability. Structural changes affected segment maintenance, emphasizing the need for precise stem-loop architecture for efficient replication. The findings provide novel insights into how FBNYV regulates genome integrity and segment functionality.