Mutational Analyses Of Genome Terminal Cis-acting Elements Of Sonchus Yellow Net Virus

The 3’-and 5’-terminal noncoding regions of nonsegmented negative RNA virus termed leader and trailer sequences, respectively, can form panhandle secondary structure and are known to contain sequences essential for directing RNA replication and transcription. Reverse genetics systems have been established for many animal negative stranded RNA (NSR) viruses since early in the 1990s. Researchers explored the functions of terminal cis-acting elements using these powerful tools and discovered these essential elements as well as stem-loop secondary structure involved in the process of transcription and replication of animal NSR viruses. Because of the lacking of reverse genetics systems in plant NSR viruses, it remains unknown about possible functions of terminal ciss-acting elements of plant NSR viruses. Sonchus yellow net virus (SYNV) is a plant nonsegmented NSR virus belonging to Nucleorhabdovirus in the Rhabdoviridae family. Our study focused on mutational analyses of the secondary structure formed by leader and trailer sequences of SYNV by means of SYNV minireplicon (SYNV-MR) and other supporting plasmids.First of all, RACE (Rapid Amplification of cDNA Ends) analyses was done to confirm that leader and trailer sequences in SYNV-MR is the same as the sequences of NCBI data base and can form panhandle secondary structure with stems and bulges. RNA folding predicts that the No.1-17 nt of leader sequence base-pair with the terminal trailer sequence to form a stem structure, where almost nucleotides are complement except the No.8 C residue and No.18-21 nt (UUUA) bulge, the No.28U bulge, among the first 30 nt of leader and trailer regions.1. Mutational analyses of the terminal 1-17 nt stem structureMutational analyses of stem indicated that the 17 nt of leader are vital to SYNV transcription. Deletion of the first 3 nt,6 nt,10 nt of leader, or the 3 nt,5 nt,10 nt of trailer all decreased reporter expression. Deleting the first 3 nt greatly reduced transcription. Further, opening basepairs in stem increasingly by mutating trailer sequence reduced SYNV mRNA transcription.After exchanging nucleotides of leader and trailer in stem, SYNV reporter genes transcription were not observed. It shows that the first 17 nt in leader sequence contains promoter for mRNA transcription and trailer sequence might involve in transcription through complementary structure.2. Mutational analyses of 18-21 nt bulgelWe made enormous mutations to bulgel and found that bulgel structure per se as well as the sequence are essential for SYNV mRNA transcription. Mutation analyses of bulgel showed that SYNV transcription remained unaffected when four nucleotides in bulgel were A or U, but greatly reduced when all four nucleotides were C or G. Interestingly, transcription was not affected with middle 2 nucleotides were CC or GG while two sides were A or U. In addition, keeping the 3 sequential nucleotides in bulgel unchanged while substituting the first or forth A/U nucleotide to C/G did not affect mRNA transcription, with the exception of UUUG mutant, which reduced SYNV mRNA transcription by nearly 50%. Other alterations to bulgel demonstrated that the transcription level of mutants in which C or G was located in side would be significantly affected, especially for the mutant GAGG.3. Mutational analyses of 28U bulge2Mutation analyses of the No.28 nt single nucleotide bulge showed this structure is also essential to SYNV transcription. When deleting 28U, or moving the bulgy U to the opposite trailer region, the reporter genes in SYNV-MR were expressed in an extremely low level. In addition, adding an A in the corresponding trailer region that could anneal to the No.28 bulgy U in the leader sequence also reduced SYNV transcriptional level. What’s more, when changing No.28 U to either A, G or C nucleotides, only mutant 28C reduced SYNV transcriptional level.In summary, our study suggested that the first 10 nt of leader and trailer equences contain transcription signal and trailer might involve in virus transcription through complement with leader sequence. Nucleotide C or G in bulges have a disadvantage for SYNV transcription. Furthermore, the bulge structures formed by first 30 nt of leader and trailer sequence may benefit recognition by RNA polymerase.