Nsp3 is an unstructured, multi-domain protein of SARS-CoV-2 and other coronaviruses. Mac3 domain of Nsp3 strongly binds guanine-rich oligonucleotide sequences of host mRNAs. Such oligonucleotides can form four-stranded structures known as G-quadruplexes, which are stabilized with hydrogen bonding of guanine residues. Mac3 domain and amino acid residues involved in binding guanine-rich oligonucleotides are conserved in SARS-CoV-2. In this work, we have synthesized three potential Mac3 target sequences, which are found in 3’ untranslated mRNA regions of the proteins TAB3 and RAB6B. Protein TAB3 has an important role in activation of NF-κβ signaling pathway, which regulates transcription of various transcription factors involved in cell growth regulation, proliferation and apoptosis in virus-infected cells. Protein RAB6B regulates the retrograde membrane transport in nervous tissue. With 1D 1H NMR experiments, we determined the ability of the oligonucleotides TAB3 6.2, RAB6B 3.3 and RAB6B 5.3 to form G-quadruplex structures. We have shown that at lower concentration of K+ ions oligonucleotides RAB6B 3.3 in TAB3 6.2 form several different G-quadruplex structures, while oligonucleotide RAB6B 5.3 formed predominantly one G-quadruplex. We performed titration with the peptide P8 of Mac3 domain of SARS-CoV-2, which has four of eight amino acid residues needed for interaction with guanine-rich sequences in SARS-CoV. With two-dimensional NMR experiments we have determined amino acid residues in P8 that are involved in the interaction with RNA molecules.
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