5' and 3' truncations of the RSE indicated that the minimal functional element is 155 nts. Truncations from the 5' and 3' end of the RSE were generated by PCR and cloned after the gag natural termination codon. Nomenclature of each construct indicates the nucleotide residue number. Diagram of deletions is to scale, with the location of the RPA probe used indicated. Each construct was transiently transfected into CEFs and RNA steady-state levels were assayed 48 hours later by RNase protection assay. Transfection efficiency was normalized using a wild-type viral loading control. RNA levels are reported as a fraction of wild-type RNA. Standard deviations are represented on the bar graph. Values represent the average of at least four experiments. Stars indicate a significant reduction compared to the wild-type virus (**: p < 0.001; ***: p < 0.0001).

Mutations within the minimal RSE suggest that multiple regions of sequence and structure contribute to function. A. Steady-state RNA levels obtained by RNase protection assay of each point mutant. Point mutations within each of the predicted stem-loops resulted in a partial loss of stability relative to the wild-type viral RNA. A compensatory mutation (Comp3) of Mutant 3 that was predicted to reform the secondary structure did not recover wild-type levels. B. Structural diagram of the predicted secondary structure of the minimal RSE based on in vitro structure studies. The regions that were deleted (Δ1 and Δ2) and the stems that were mutated (Mut1-Mut3) are indicated. The boxes corresponding to each mutation indicate the sequence variation (highlighted region) while the structure displays the wild-type sequence. C. A 14 nucleotide (deletion 1) and a 12 nucleotide (deletion 2) single-stranded region of the minimal RSE were deleted and the level of viral RNA was assayed by RNase protection assay. Values are reported as a fraction of wild-type RNA. The deletions resulted in a partial loss of function. To determine whether spacing within the RNA was altered, sequence was added to the 5' or 3' end or at the deletion site. D. Diagram depicting the organization of the deletion constructs and the location of the sequence added back. Δ: The location of the deleted sequence is represented by a dotted line. Mix: The checked box represents deleted sequence that was scrambled and then added back at the deletion site. 5': The diagonally hatched box represents 10 nts of viral sequence added back to the 5' end of the minimal RSE sequence that contains the deletion. 3': The diagonally hatched box represents 10 nts of viral sequence added back to the 3' end of the minimal RSE sequence that contains the deletion. The added viral sequence is that which naturally lies just 5' or just 3' of the minimal RSE. The diagram is not to scale. Stars indicate a significant reduction compared to the wild-type virus (*: p < 0.01; **: p < 0.001; ***: p < 0.0001)

The immediate termination codon context did not affect levels of the unspliced viral RNA. A. The fourth nucleotide of the termination codon signal was altered to each of the four possible ribonucleotides. This was done at the gag stop codon, with and without the RSE and at a premature termination codon at nucleotide 1250. Steady-state levels of RNA were assayed by RNase protection assay. These mutations did not significantly affect the stability of the wild-type viral RNA (WT gag UAG), nor the efficiency of decay of the RNAs bearing a premature termination codon within gag (PTC UAG) or lacking the RSE (ΔRSE UAG). B. The stop codon of the gag termination codon (UAG) was altered to each of the other two stop codons (UAA and UGA). The level of steady-state viral RNA was not affected, as assayed by RNase protection assay. Representative RNase protections are shown. Stars indicate a significant reduction compared to the wild-type virus (**: p < 0.001; ***: p < 0.0001)

The minimal RSE functions only after the gag termination codon. The RSE was cloned in the forward (2660-2880 For) and reverse orientation (2660-2880 Rev) after a premature termination codon within gag at position 1250. The minimal RSE (2577-2732) and a slightly longer RSE fragment (2567-2732) were cloned after the same premature termination codon. 2660-2880 For is a previously described functional fragment of the RSE (Cfor; [25]). The minimal RSE is unable to stabilize the RNA when placed after a premature termination codon within gag. RNA levels were assayed by RNase protection assay. Representative gels are shown below each graph. Stars indicate a significant reduction compared to the wild-type virus (***: p < 0.0001)

The minimal RSE protected the gag stop codon from recognition by the NMD machinery. Co-transfection of wildtype, ΔRSE, 2577-2732 (minimal RSE) and 2788-2732 with wildtype Upf1 or a dominant negative form of Upf1 (RR857GA). Each construct was transiently transfected into CEFs, and RNA steady-state levels were assayed 48 hours later by RNase protection assay. A representative RNase protection assay is shown, with the set of bands below each bar corresponding to the construct indicated directly above on the graph. The top band of the gel (experimental) is a fragment of gag probe protected during the RNase protection assay corresponding to the unspliced viral RNA from the experimental construct. The bottom band (control) is a wild-type viral loading control that protects a different sized fragment of the same gag probe due to a small deletion. Standard deviations are represented on the bar graph. Values represent the average of at least four experiments. A star indicates a significant reduction compared to the corresponding viral construct co-transfected with wild-type Upf1 (*: p < 0.01).

A premature termination codon inserted within the RSE at position 2685 underwent decay when readthrough of the gag stop codon was forced. A. Schematic of stop codon locations within the RSE. Premature termination codons were generated within the RSE at positions 2535, 2586, 2631, 2685 and 2736, named 1-5, respectively. The black font represents the minimal RSE as determined by truncations. Text in grey is RSE sequence flanking the minimal RSE. B. The RSE containing these mutations was cloned into two constructs, one with the natural gag termination codon sequence (WT gag stop) and the other with a single nucleotide insertion preceding the gag termination codon that forced readthrough into the pol open reading frame (Readthrough gag stop). Only PTC4 at position 2685 underwent decay in the readthrough construct. A representative RNase protection assay is shown, with the set of bands below each bar corresponding to the construct indicated directly above on the graph. Stars indicate a significant reduction compared to the wild-type virus (***: p < 0.0001)

The natural viral UTRs can substitute for the RSE after the gag termination codon, but not after a premature termination codon. A. Diagram of the viral UTR cloning strategy. The UTRs of pol, env, and src (black arrows) were cloned after the gag natural stop codon (grey octagon) and after a premature termination codon (white octagon) within gag at position 1250. B. The pol and src UTRs were able to maintain wild-type RNA levels when placed after the gag natural termination codon. None of the UTRs other than the RSE was capable of stabilizing the RNA when placed after a premature termination codon. RNA levels were assayed by RNase protection assay. Representative gels are shown below each graph. Stars indicate a significant reduction compared to the wild-type virus (**: p < 0.001; ***: p < 0.0001)

The RSE may act as an NMD insulator. Either through a direct interaction with the RNA, or mediated through a protein, the RSE may act as an NMD insulator. A. The RSE may act as a decoy to conceal the gag stop codon from association with some NMD factors or to interact with Smg8/9 to keep Smg1 inactive. B. The RSE may interact with the translation termination machinery at a site that overlaps with that of Upf1, such that the NMD machinery can no longer associate with eRF3. C. The RSE may interact with eRF3 to promote translation termination. D. The RSE may interact directly with one of the ribosomal subunits to induce a conformational change that favors translation termination.