Epistemic_Statements  

CORD-19:0cf35bb67d4a9d57d3482d3acc790525eab934eb JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 0-71 Epistemic_statement denotes Structure and Dynamics of the HIV-1 Frameshift Element RNA Terms of Use
T2 82-235 Epistemic_statement denotes The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target.
T3 326-425 Epistemic_statement denotes Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome.
T4 547-659 Epistemic_statement denotes These structural elements also support the presence of a secondary frameshift site within the frameshift domain.
T5 811-929 Epistemic_statement denotes Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain.
T6 930-1034 Epistemic_statement denotes Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation.
T7 1203-1375 Epistemic_statement denotes These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs.
T8 1521-1708 Epistemic_statement denotes Although the Gag and Pol polyproteins are translated together, the pol gene is encoded in a reading frame offset from the upstream gag reading frame by one nucleotide in the 5′ direction.
T9 2212-2333 Epistemic_statement denotes This downstream structure is thought to pause the ribosome while the A and P sites are occupied by the slippery sequence.
T10 2334-2549 Epistemic_statement denotes Although the precise mechanism is not fully understood, frameshifting occurs with a frequency of 5−10% in cultured HIV-transfected human cells, and the Gag to Gag-Pol ratio appears to be important for viral fitness.
T11 2550-2668 Epistemic_statement denotes 3 The frameshifting process has consequently attracted interest as a target for the development of therapeutic agents.
T12 2755-2954 Epistemic_statement denotes 6 A number of refinements and extensions of this model have been proposed that include additional sequence and structures including pseudoknots, 7−9 a triple-stranded RNA species, 10 and two helices.
T13 3814-3964 Epistemic_statement denotes 16 SHAPE reactivities can be incorporated into thermodynamics-based folding algorithms 17 resulting in highly accurate RNA secondary structure models.
T14 4258-4395 Epistemic_statement denotes SHAPE data also support the formation of three helices outside the domain traditionally identified as the frameshift stimulatory element.
T15 4522-4713 Epistemic_statement denotes Finally, low SHAPE reactivity at a 3-nt strand between the alternate lower stem and anchoring helix supports an additional short secondary structure, which we currently model as a 3-bp helix.
T16 4714-4858 Epistemic_statement denotes SHAPE data suggest that the frameshift domain spans 140 nts, a significantly larger region than is included in the two-helix model ( Figure 1 ).
T17 5440-5746 Epistemic_statement denotes 22 Our results provide strong support for the SHAPE-directed frameshift model, and the functional importance of SHAPE-detected conformations for frameshifting reveals that the frameshift domain is a dynamic element capable of structural remodeling and supports the existence of a secondary frameshift site.
T18 8909-9048 Epistemic_statement denotes When possible, we included flanking single-stranded regions as part of the targeted strand to facilitate LNA binding to structured helices.
T19 11024-11219 Epistemic_statement denotes The resulting reactivities span a scale from 0 to ∼1.5, where 0 indicates no reactivity (and a highly constrained nucleotide) and reactivities >0.7 typically indicate highly flexible nucleotides.
T20 14657-14728 Epistemic_statement denotes LNA Binding Supports the SHAPE-Directed Model of the Frameshift Region.
T21 14839-14988 Epistemic_statement denotes However, addition of LNAs directed against the anchoring helix failed to produce detectable SHAPE reactivity changes in the predicted partner strand.
T22 14989-15150 Epistemic_statement denotes This result could reflect either that the targeted helix was too stable to be disrupted by LNA binding or that our model in the frameshift region was inaccurate.
T23 16472-16654 Epistemic_statement denotes If the targeted helix existed and if LNA binding disrupted native base pairing, we expected to observe increased SHAPE reactivity at the strand complementary to the LNA-bound strand.
T24 16787-16974 Epistemic_statement denotes These results strongly support the formation of the slippery sequence (Figure 2A,B) and alternate lower ( Figure 2C ,D) and anchoring helices ( Figure 2E ,F) in the full-length HIV-1 RNA.
T25 17678-17874 Epistemic_statement denotes The changes in SHAPE reactivity upon LNA binding suggest the formation of base-pairing interactions that correspond to the lower stem from the conventional two-helix model (Figure 2A, red boxes) .
T26 17991-18099 Epistemic_statement denotes LNA 2 targets nucleotides in close proximity to, and possibly overlapping with, the conventional lower stem.
T27 18100-18210 Epistemic_statement denotes In this case, steric occlusion due to LNA 2 binding likely disfavors formation of the conventional lower stem.
T28 18831-19011 Epistemic_statement denotes We attribute this lack of a conformational switch to the fact that LNA 4 binding would prevent the formation of both the alternate and conventional conformations of the lower stem.
T29 19460-19543 Epistemic_statement denotes These results suggest that the anchoring helix stabilizes the alternate lower stem.
T30 20259-20384 Epistemic_statement denotes These results suggest that the alternate lower stem and slippery sequence stabilize one another and are structurally coupled.
T31 21144-21343 Epistemic_statement denotes Nucleotides involved in the alternate lower stem exhibited reactivity changes that suggest a switch to the conventional lower stem at 20% formamide ( Figure 3B and Supporting Information, Figure 2 ).
T32 21344-21451 Epistemic_statement denotes In 40% formamide, SHAPE reactivities indicated that the conventional lower stem was unfolded ( Figure 3C ).
T33 21452-21655 Epistemic_statement denotes Nearest neighbor thermodynamic calculations indicate that the conventional lower stem is slightly more stable than the SHAPEdirected alternate lower stem (−8.4 kcal/mol and −8.2 kcal/ mol, respectively).
T34 21656-21948 Epistemic_statement denotes We hypothesize that the formation of the alternate lower stem is largely dependent on stabilization from other elements in the domain and that, when these interactions are disrupted by LNA binding or by formamide denaturation, the slightly more stable conventional lower stem is able to form.
T35 22755-22872 Epistemic_statement denotes Structural interrogation of the in virio state is possible because SHAPE reagents readily cross biological membranes.
T36 23303-23396 Epistemic_statement denotes These differences could reflect RNA refolding or protein binding in the in virio environment.
T37 23397-23542 Epistemic_statement denotes In contrast, SHAPE reactivities for the anchoring helix and upper stem remained low, suggesting that these elements folded stably inside virions.
T38 24280-24410 Epistemic_statement denotes However, when the alternate lower stem was disrupted, a significant decrease in frameshifting was observed ( Figure 5, mutant 4) .
T39 24560-24646 Epistemic_statement denotes These results suggest that the alternate lower stem is a potential therapeutic target.
T40 25136-25297 Epistemic_statement denotes 22 Together, these data are consistent with a translational pausing mechanism whereby structured regions preceding the frameshift site regulate ribosome spacing.
T41 25298-25608 Epistemic_statement denotes 37 That these results differ from those obtained when the anchoring helix and alternate lower stem were mutated independently supports our hypotheses that there is strong structural coupling between these elements and that these helices have overlapping functional roles in modulating frameshifting efficiency.
T42 25695-25832 Epistemic_statement denotes The alternate lower stem overlaps with a UUUUCUU sequence (nucleotides 1676−1682) that resembles the conserved UUUUUUA slippery sequence.
T43 25938-26085 Epistemic_statement denotes 38 At the protein level, this causes a Leu to Phe mutation that may increase cleavage efficiency and enhance the production of functional protease.
T44 26086-26325 Epistemic_statement denotes This site could also act as a secondary slippery sequence that increases the overall amount of frameshifting, thereby increasing the relative amount of protease to compensate for reduction in protease activity in the presence of inhibitor.
T45 26704-26862 Epistemic_statement denotes 40 However, the construct used could not form flanking structures such as the anchoring helix and the adjacent 3-bp helix predicted by SHAPE-directed folding.
T46 27991-28160 Epistemic_statement denotes Our data provide support for the three main helices unique to the frameshift domain model proposed based on SHAPE-directed folding of an entire HIV-1 genome (Figure 1 ).
T47 28161-28444 Epistemic_statement denotes 15 Models for the frameshift domain that contain pseudoknots have recently been proposed; 9 however, ex virio SHAPE data ( Figure 1 ) and prior frameshifting assays 22 do not support a contribution of these pseudoknots to the observed structural ensemble or frameshifting efficiency.
T48 28445-28564 Epistemic_statement denotes Our model shares a stable upper stem with the previously proposed two-helix model 11 but has two important differences.
T49 28565-28662 Epistemic_statement denotes First, this study supports alternative pairing partners for bases in the conventional lower stem.
T50 29417-29677 Epistemic_statement denotes Two of these helices, the alternate lower stem and the helix that sequesters the slippery sequence, appear to mutually stabilize each other because disruption of either of these helices by LNA binding destabilized or induced refolding of the other (Figure 2) .
T51 29678-29752 Epistemic_statement denotes Additionally, the anchoring helix likely stabilizes both of these helices.
T52 29753-29919 Epistemic_statement denotes The frameshift domain adopts the same structure whether the genomic RNA is probed directly after extraction from virions or is first heat-denatured and then refolded.
T53 29920-30159 Epistemic_statement denotes This similarity suggests that the differences between the SHAPE-directed model and the conventional model are due primarily to the presence of the complete sequence of the viral RNA that was used in development of the SHAPE-directed model.
T54 30264-30489 Epistemic_statement denotes However, inside virions, portions of the frameshift domain are less structured than in the extracted RNA, suggesting that higher-order protein or ligand interactions modulate domain structure in the native virion environment.
T55 30765-31063 Epistemic_statement denotes In the absence of tertiary structure or other potentially stabilizing interactions, the conventional lower stem is predicted to be slightly more stable than the SHAPE-directed alternate lower stem, and in 20% formamide, we observed a switch from the alternative stem to the conventional lower stem.
T56 31064-31170 Epistemic_statement denotes These data suggest that the structure of the frameshift region is dependent on the local microenvironment.
T57 31171-31311 Epistemic_statement denotes The changes induced by LNA binding suggest that the structure of the frameshift domain is dynamic as the ribosome moves through this region.
T58 31451-31587 Epistemic_statement denotes LNA binding experiments indicate that unwinding of this helix induces a major structural rearrangement throughout the frameshift domain.
T59 31739-31900 Epistemic_statement denotes The functional implications of this structural rearrangement are not understood, but the Construct Δss has mutations that disrupt the standard slippery sequence.
T60 31953-32059 Epistemic_statement denotes Construct 6 additionally includes a C-to-U mutation that creates the proposed secondary slippery sequence.
T61 32380-32638 Epistemic_statement denotes 41 Prior studies using an RNA containing up to 90 nucleotides of the HIV-1 frameshift domain sequence indicate that sequences in the conventional lower stem are functionally important for frameshifting in cultured cells but not in rabbit reticulocyte lysate.
T62 33596-33749 Epistemic_statement denotes LNA 2 binding data suggest that unwinding of the conventional lower stem results in a switch back to the alternate lower stem conformation ( Figure 7C ).
T63 33750-33833 Epistemic_statement denotes The ribosome must then unwind the very stable upper stem and switch reading frames.
T64 33834-33961 Epistemic_statement denotes The high stability of the anchoring helix suggests that this structure may reform after the ribosome has translated through it.
T65 33962-34101 Epistemic_statement denotes If this is the case, the ribosome would encounter the anchoring helix for a second time before exiting the frameshift domain ( Figure 7D ).
T66 34102-34285 Epistemic_statement denotes The adjacent 3-bp helix could also potentially reform and would be able to serve as the frameshift stimulatory stem in HIV-1 sequence variants that contain a second slippery sequence.
T67 34286-34424 Epistemic_statement denotes Frameshifting efficiency assays performed in the present study revealed that the secondary slippery site can indeed promote frameshifting.
T68 34923-35218 Epistemic_statement denotes 22 Consistent with this eight nucleotide distance, our results indicate that frameshifting from the second slippery site is dependent upon the integrity of the 3-bp helix but not the anchoring stem, which would be too far away from the second slippery site to induce frameshifting ( Figure 6 ).
T69 35427-35665 Epistemic_statement denotes 15 The frameshift region has significant conformational flexibility, and we hypothesize that a switch occurs between the SHAPE-predicted helices and the two helices in the conventional model as the ribosome unwinds the frameshift element.
T70 35666-35874 Epistemic_statement denotes From a functional perspective, the alternate lower stem is important for native-like frameshift levels in the context of the larger domain structure and consequently represents a potential therapeutic target.
T71 35875-36128 Epistemic_statement denotes We also examined the sequence and structural requirements of a previously identified secondary frameshift site and found that its function is dependent on structural elements outside the traditional frameshift domain but within the SHAPE-directed model.