Cis-acting regulatory elements in the potato virus X 3' non-translated region differentially affect minus-strand and plus-strand RNA accumulation.

Abstract The 72 nt 3′ non-translated region (NTR) of potato virus X (PVX) RNA is identical in all sequenced PVX strains and contains sequences that are conserved among all potexviruses. Computer folding of the 3′ NTR sequence predicted three stem-loop structures (SL1, SL2, and SL3 in the 3′ to 5′ direction), which generally were supported by solution structure analyses. The importance of these sequence and/or structural elements to PVX RNA accumulation was further analyzed by inoculation of Nicotiana tabacum (NT-1) protoplasts with PVX transcripts containing mutations in the 3′ NTR. Analyses of RNA accumulation by S 1 nuclease protection indicated that multiple sequence elements throughout the 3′ NTR were important for minus-strand RNA accumulation. Formation of SL3 was required for accumulation of minus-strand RNA, whereas SL1 and SL2 formation were less important. However, sequences within all of these predicted structures were required for minus-strand RNA accumulation, including a conserved hexanucleotide sequence element in the loop of SL3, and the CU nucleotide in a U-rich sequence within SL2. In contrast, 13 nucleotides that were predicted to reside in SL1 could be deleted without any significant reduction in minus or plus-strand RNA levels. Potential polyadenylation signals (near upstream elements; NUEs) in the 3′ NTR of PVX RNA were more important for plus-strand RNA accumulation than for minus-strand RNA accumulation. In addition, one of these NUEs overlapped with other sequence required for optimal minus-strand RNA levels. These data indicate that the PVX 3′ NTR contains multiple, overlapping elements that influence accumulation of both minus and plus-strand RNA.