Summary auto-generated
This study characterizes the sequence requirements for viable recombinant formation between two tobraviruses: tobacco rattle virus (TRV) and pea early browning virus (PEBV). Researchers constructed hybrid cDNA clones by combining RNA2 segments from both viruses with swapped 5' or 3' non-coding regions. Testing these hybrids with homologous and heterologous RNA1 replicases revealed that the 5' non-coding region (NCR) determines template specificity. TRV RNA1 successfully replicated RNA2 containing TRV's 335 nucleotides at the 5' terminus, regardless of whether the 3' terminus came from PEBV. Similarly, PEBV RNA1 replicated RNA2 with PEBV's 320 nucleotide 5' terminus, even with TRV 3' sequences. In contrast, swapping only 3' NCRs permitted cross-replication. In vitro translation experiments using PEBV transcripts with 5' non-coding deletions confirmed that coat protein is expressed from a subgenomic RNA and that heterologous viral replicases can recognize the coat protein subgenomic promoter. These findings explain how natural recombinant isolates can form and establish that 5' non-coding sequences, not 3' regions, govern viral RNA replication specificity in tobraviruses.
Key findings
- The 5' non-coding region of tobravirus RNA2 determines replicase template specificity; 320-335 nucleotides from the 5' terminus are sufficient for recognition by homologous RNA1
- The 3' non-coding region does not determine template specificity; RNA2 with heterologous 3' termini are replicated by cognate replicases
- PEBV RNA1 shows occasional low-level replication of RNA2 with heterologous TRV 5' sequences, while TRV RNA1 cannot replicate RNA2 with PEBV 5' sequences
- Tobravirus coat protein is expressed from a subgenomic RNA controlled by a promoter in the 5' non-coding region
- Viral replicases can recognize and utilize coat protein subgenomic promoters from heterologous viruses, explaining natural recombinant formation
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Abstract
Natural recombinant tobacco rattle tobravirus (TRV) isolates contain sequences from a different tobravirus, pea early browning virus (PEBV). To characterize the sequence requirements for viable recombinant formation hybrid cDNA clones of RNA2 of PEBV and TRV were assembled. Inclusion of 320 nt from the 5' terminus of PEBV or 335 nt from the 5' terminus of TRV in the hybrid RNAs was sufficient to permit their replication by, respectively, PEBV RNA1 or TRV RNA1 regardless of the origin of the 3' terminal region. However, PEBV RNA1 but not TRV RNA1 was sometimes able to support low level replication of RNA2 containing the heterologous 5' terminal region. In vitro translation of PEBV transcripts containing 5' noncoding region deletions supported the hypothesis that in vivo the PEBV coat protein (CP) is expressed from a subgenomic RNA and that, therefore, in the recombinants the CP subgenomic promoter probably is recognized by the replicase of the heterologous virus.