Structure-altering mutations of the SARS-CoV-2 frameshifting RNA element

Abstract

With the rapid rate of Covid-19 infections and deaths, treatments and cures besides hand washing, social distancing, masks, isolation, and quarantines are urgently needed. The treatments and vaccines rely on the basic biophysics of the complex viral apparatus. While proteins are serving as main drug and vaccine targets, therapeutic approaches targeting the 30,000 nucleotide RNA viral genome form important complementary approaches. Indeed, the high conservation of the viral genome, its close evolutionary relationship to other viruses, and the rise of gene editing and RNA-based vaccines all argue for a focus on the RNA agent itself. One of the key steps in the viral replication cycle inside host cells is the ribosomal frameshifting required for translation of overlapping open reading frames. The RNA frameshifting element (FSE), one of three highly conserved regions of coronaviruses, is believed to include a pseudoknot considered essential for this ribosomal switching. In this work, we apply our graph-theory-based framework for representing RNA secondary structures, “RAG” (RNA-As Graphs), to alter key structural features of the FSE of the SARS-CoV-2 virus. Specifically, using RAG machinery of genetic algorithms for inverse folding adapted for RNA structures with pseudoknots, we computationally predict minimal mutations that destroy a structurally-important stem and/or the pseudoknot of the FSE, potentially dismantling the virus against translation of the polyproteins. Our microsecond molecular dynamics simulations of mutant structures indicate relatively stable secondary structures. These findings not only advance our computational design of RNAs containing pseudoknots; they pinpoint to key residues of the SARS-CoV-2 virus as targets for anti-viral drugs and gene editing approaches.

To knot or not to knot: Multiple conformations of the SARS-CoV-2 frameshifting RNA element

“…Length-dependent predictions for the FSE were already presented in our prior work. 20 Here we show further predictions with associated graph topologies in Fig. 3 using PKNOTS, NUPACK, IPknot, and ProbKnot.…”

“…The darker purple shadings indicate greater sequence homology. Based on our work presented in 20 and here, we annotate Stems 1 and 3 of SARS-CoV-2 FSE in black, and Stem 2 corresponding to the H-type pseudoknot (dual graph 3_6) in red, the HL-type pseudoknot (3_3) in green, and the 3-way junction (3_5) in purple.…”

“…Because the unknotted 3-way junction (3_5) also emerges as a minor player in the 77nt FSE conformational landscape, we obtain reactivity data for the double mutant we had predicted in our prior work 20 to stabilize this fold over the two pseudoknots. Fig.…”

“… 17 , 18 , 42 Since SARS-CoV-2 FSE only differs in one base, many studies presume that this FSE will continue to adopt the 3_6 conformation. Using various techniques and sequence lengths, 10 out of the 16 papers show a major 3_6 pseudoknot: iterative 2D prediction for 68nt; 43 3D modeling and MD simulation for 68nt; 44 2D prediction, NMR, DMS footprinting for 69nt; 45 2D, 3D, and MD simulation for 77nt and 84nt; 20 small-angle X-ray scattering for 85nt; 19 DMS-MaPseq for 85nt; 22 homology model for 88nt; 46 Cryo-EM for 88nt 23 and 118nt; 21 deep sequencing for 1475nt. 47 All except the last two studies use short FSE lengths of 68–88nt.…”

“…We also noted that structural predictions of the FSE by available programs depend on the length considered, 20 in particular, whether the slippery site is included. This inclusion is both computationally and biologically meaningful, since the slippery site is thought to be inaccessible while the FSE is in direct interaction with the ribosome, but possibly free otherwise.…”