Identification of conserved three-way junction in the genome of the bovine foamy virus

Three-way junctions (3WJs) belong to unusual structures in DNA and RNA. 3WJs are non-canonical structures like G-quadruplexes, triplexes (H-DNA), cruciform, hairpin structures, A-DNA, and Z-DNA that differ from the classic double-stranded B-DNA. 3WJs play an important role in many biological processes and may be associated with some human diseases. This study aimed to search for putative 3WJ structures in the mRNA of bovine foamy virus (BFV). Bioinformatic analysis was used to analyze conserved RNA structural motifs of intramolecular 3WJ in BFV mRNA. The Vfold2D software was used to search for structural motifs in the 3WJ RNA. Multiple sequence alignment was conducted using MEGA software. For the confirmation of secondary structures and the determination of the thermodynamic parameters of 3WJs, Mfold software from the UNAFold web server was utilized. Based on multiple alignments of 37 BFV isolates with the complete genome, we found 6 putative 3WJ structures in the BFV mRNA, which are stabilized by 20–26 complementary nucleotides pairs (ntp) and localized in the gag, env, bel2 genes, as well as in the 5’LTR. However, only two 3WJ structures in gag and env genes from the abovementioned six ones, designed by the Mfold software, coincide with 3WJ structures determined by the Vfold2D software. Five 3WJ structures from 6 identified ones are not conserved. Conserved 3WJ structure with a length of 73 nt for a set of 37 BFV isolates with complete genome is localized between 5’-LTR and 5’-end of gag gene and partially covers 5’-end of gag gene. This intramolecular secondary structure is formed by three duplexes and stabilized by 20 complementary ntp with a free energy of −19.8 kcal/mol. Our analysis of SNPs in the paper (Bao et al., 2020), which arose after serial passages of BFV Riems-infected MDBK cells has shown that the determined 3WJ structure is retained, indicating the importance of this alternative structure for BFV functioning