Giant grouper nervous necrosis virus subgenomic RNA3 is transcribed by a premature termination mechanism via intragenomic RNA-RNA pairing.

BACKGROUND: Nervous necrosis virus (NNV) is a bipartite positive-sense RNA virus that causes viral nervous necrosis disease in larval and juvenile marine fish. In its genome, the RNA1 segment encodes RNA-dependent RNA polymerase (RdRp) for viral RNA synthesis, while RNA2 encodes coat protein for viral particle assembly. In addition, an RNA1-derived subgenomic RNA3 region encodes the non-structural B2 protein, which antagonizes host RNA interference mechanisms. However, the mechanisms of RNA3 transcription remain unclear. METHODS: A DNA-based reverse genetic platform vector (pCMV-DRz) for recombinant (r) giant grouper (GG) NNV recovery was constructed. End sequences of GGNNV RNAs were determined by 5' rapid amplification of cDNA ends (RACE) and/or circular RNA sequencing approach. Replication and transcription of viral RNAs in GGNNV-infected or mutant clones transfected grouper brain (GB) cells were detected by Northern blotting using anti-sense RNA DIG-labeling probes. Recombinant viral proteins were detected by using Western blotting or immunocytochemistry staining after GB cells transfected with pCMV-DRz-RNA1 or infected with rGGNNV. In addition, the viral kinetics of rGGNNV was determined using TCID(50) assay. Finally, the minimum free energy secondary RNA structures were predicted using RNA-Fold of Vienna RNA Websuite. RESULTS: In the reverse genetic platform, high viral titer of rGGNNV (10(8) TCID(50)/mL) was readily obtained from culture medium of pCMV-DRz-RNA1 and pCMV-DRz-RNA2 co-transfected GB cells, and the resulting genomic sequences at the viral 5'- and 3'-ends were confirmed to be accurate. The pCMV-DRz-RNA1 autonomous replicon alone (encodes RdRp) was sufficient to replicate RNA1 and produce RNA3. Without altering the encoded RdRp amino acid sequence, we performed mutagenesis of the RNA1 sequence and found that RNA3 is produced by a premature termination mechanism that involves an 8-base paired helix formed by a long-distance intragenomic RNA-RNA interaction. Furthermore, transcriptional promoter elements near the RNA3 negative-strand 3'-end also contributed to RNA1 and RNA3 production, and the expression levels of RNA1 and RNA3 were modulated by RNA2 co-expression. CONCLUSION: Our results suggest that NNV utilizes premature termination mechanism for subgenome RNA3 transcription and NNV RNAs expression is coordinately regulated by viral cis- and trans-RNA sequences.