Cis-acting Regulatory Elements in the Potato Virus X 3′ Non-translated Region Differentially Affect Minus-strand and Plus-strand RNA Accumulation

Abstract

The 72nt 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.

Potato virus X: a model system for virus replication, movement and gene expression

“…Because minus‐strand synthesis initiates from the 3′‐terminus of the genome, it is likely that sequences and/or structures in the 3′‐region are required for template recognition by the viral replicase complex and for the initiation of RNA synthesis. All PVX strains sequenced to date have identical sequences in the 74 nt 3′‐NTR (Pillai‐Nair et al ., 2003), suggesting that the sequence in this region is important for virus viability. Analyses of computer‐predicted structures and solution structural probing indicate that the PVX 3′‐NTR can fold into three stem‐loop structures, but the formation of only one (SL3; Fig.…”

3′ RNA Elements in Hepatitis C Virus Replication: Kissing Partners and Long Poly(U)

“…Several viral and cellular mRNAs contain a short U-rich region upstream of the poly(A) signal. This sequence recruits accessory proteins, such as hFip1, PTB, Hu family proteins, cleavage and polyadenylation specificity factor components, and U2AF 65 , which facilitate polyadenylation and complete the formation of the mRNA 3Ј end (1,11,17,21,23,35,52). In addition, the U-rich region of the myosin phosphatase targeting subunit 1 pre-mRNA has been shown to mediate an alternative splicing event by interacting with either T-cell intracellular antigen 1 or PTB during tissue-specific developmental regulation (37).…”

Identification of the Capsid Protein-binding Region of the SL1(+) RNA Located at the 5' Region of the Potato virus X Genome

“…By conducting EMSA, UV cross-linking, and yeast three-hybrid analyses, we identified those sequences and/or regions of the PVX SL1(+) RNA that are required for CP binding. The SL structures at the 5' end of the positive-strand RNA of the genomes contain at least three well-defined stem-loop structures (SL1, SL2, and SL3 in the 3' to 5' direction) and are well conserved in most reported potexviruses (Chen et al, 2010;Miller et al, 1998;Pillai-Nair et al, 2003). In this regard, it is worth noting that SL1(+) structures are identical in all sequenced PVX strains.…”

“…Previous studies have indicated that the 5' and 3'-terminal nt of the PVX genome can form conserved SL structures (Miller et al, 1998;Pillai-Nair et al, 2003). The SL structure formed by the 5'-terminal nt containing the ACrich single-stranded region and the SL1 secondary structure is more stable than that formed by the 3'-terminal nt.…”