Z-RNA and the Flipside of the SARS Nsp13 Helicase: Is There a Role for Flipons in Coronavirus-Induced Pathology?

Figure 1. Coronaviruses and human host susceptibility (A) Coronaviruses (CoV) with the AACGAAC transcription regulatory sequence (TRS) and red labels are associated with severe disease in humans. This TRS is present in CoV from other species but not in all human CoV, especially those that cause mild disease (B) Host responses to double-stranded Z-RNA viruses can produce inflammatory cell death through the activation of Receptor Interacting Protein Kinases (RIPK) by Z-DNA binding Protein q (ZBP1). The interaction between RIPK and ZBP1 is through their RIP Homotypic Interaction Motifs (RHIM). (C) Dimers formed from each RHIM protein stack on each other to form a fiber that provides a scaffold for the host response machinery to assemble. The interaction of RIPK1 and RIPK3 is shown with the sequences contributing to the hydrophobic core highlighted in yellow. Blue highlights other residues that stabilize the core. The central figure shows a space-fill representation of one RIPK3 chain present in the dimer.

Figure 2. Coronavirus signatures (Z-sigs) based on Z-flipons (A) Z-sigs for each human pathogenic virus are unique. (B) Z-sigs match those for the presumed natural hosts for each virus. (C) The Z-Sigs differ from those for other known coronaviruses (D) The Z-sigs also vary with the host animal infected. The Z-sig was derived using the ZHUNT3 (ZH3) program (15) that scores the propensity of sequences to flip to the left-handed helical Z-conformation. Sequences with a score greater than 600 are likely to adopt the Z-conformation under physiological conditions. Sequences were aligned using MAFFT prior to analysis (31).

Figure 3. Regions with high ZH3-scores (>600) (A) The multiple sequence alignment of coronaviruses to SARS-COV2 genome shows potential Z-forming elements in a number of regions of the coronavirus genome. Those encoding Nsp1, Nsp2 and Nsp3 and N proteins contain highly variable regions, while those for the RNA-dependent RNA polymerase (RdRp, Nsp12), the Nsp13 helicase (inside the red box) and the Nsp14 nuclease/methyl-transferase show greater constraint. Lower case letters indicate bases that break the pyrimidine/purine alternation. The amino acid translation of the first and longest nucleotide sequence with a yellow background are highlighted with a blue background. Nucleotide positions are numbered by the SARS-COV2 genome. (B) Compared to SARS1, the helicase of SARS-CoV2 contains three adjacent non-synonymous mutations that conserve the Z-RNA stem and the peptide sequence, but diminish its Z-forming potential. The equivalent MERS sequence has a very low Z-forming potential and encodes a peptide with a lysine (K) in place of an arginine (R). A potential MERS Z-RNA prone element is close-by (within the dashed box).

Figure 4. RIP homotypic Interaction motif. (A) CoV Nsp13 protein sequences from different strains that contain the RHIM (I/V)Q(I/L/V)G residues (in yellow) were aligned with known RHIMs from other viral and host proteins (labels in the gray box). SMG1 was identified here but is not yet a validated RHIM. Residues are colored as in Figure 1. (B) Phylogram to show the sequence variation between proteins as a proportion. (HHV, human herpes virus; CMV, cytomegalovirus; ZBP1, Z-DNA binding Protein 1; RIPK, Receptor Interacting Protein Kinase; TRIF, Toll like Receptor Adaptor Molecule; SMG, SMG1 nonsense mediated mRNA decay associated PI3K related kinase; h, human; m, mouse; r, rat; t, tupaiid).

Figure 5. The interaction between the Nsp13 RHIM (residues 193-6) and PARAR (residues 335-9) are conformation dependent. (A) In the ATP-free apo form of Nsp13, the RHIM (yellow space fill carbons) contacts PARAR (crimson space fill carbons) (B) In the active state, RHIM and PARAR separate, opening the 5´ end of the single-strand RNA channel, that has the 3´end marked by N516 (white space fill carbons). (C) In the open complex, the RHIM separates further from the RecA2 domains to create a cavity that is potentially large enough to accommodate dsRNA. This opening is associated with a rotation of the Zinc binding domain (ZincBD) relative to N516. PARAR also rotates, changing the position of R337 and R339. The structures are from PDB files 7NIO (44), 6XEZ (45) and 7RDX (46) as labeled, with images rendered using the NGLViewer (47).

Figure 6. Nsp13 opens to expose a hook and platen for binding dsRNA. (A) The arginine rich PARAR hook (crimson carbons) and the tyrosine (Y205, grey carbons) platen create a surface for docking dsRNA. Tryptophan (W168)(blue carbons) has the potential to orientate Y205 through and edge to face interaction and create a Z-RNA specific recognition element like that present in the Zα domain that confers specificity for binding to the left-handed helix (48). In this open orientation, the RHIM (yellow carbons) is free to engage other RHIM proteins. The space fill with white carbons show the residues identified as making base-specific contacts with single-stranded RNA (ssRNA) in the active conformation of Figure 5B (residues 178, 179, 230, 233 in domain 1B, residues 311, 335,361, 362:E,363, 390, 408, 410:E in RecA1). (B) The view of the Nsp13 molecule from the opposite side to that shown in the first panel. (C) In addition to the ATP-dependent helicase activity, Nsp13 has the potential to capture ssRNA when Z-RNA flips to and from A-RNA. The higher energy Z-RNA powers the strand-separation, providing the DG needed to fuel the Z-flipon helicase activity we refer to as a Z-flipase.