Identification of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2 and their implications on its protein structure

Abstract

The rapid development of the SARS-CoV-2 mediated COVID-19 pandemic has been the cause of significant health concern, highlighting the immediate need for effective antivirals. SARS-CoV-2 is an RNA virus that has an inherently high mutation rate. These mutations drive viral evolution and genome variability, thereby facilitating viruses to have rapid antigenic shifting to evade host immunity and to develop drug resistance. Viral RNA-dependent RNA polymerases (RdRp) perform viral genome duplication and RNA synthesis. Therefore, we compared the available RdRp sequences of SARS-CoV-2 from Indian isolates and the 'Wuhan wet sea food market virus' sequence to identify, if any, variation between them. Our data revealed the occurrence of seven mutations in Indian isolates of SARS-CoV-2. The secondary structure prediction analysis of these seven mutations shows that three of them cause alteration in the structure of RdRp. Furthermore, we did protein modelling studies to show that these mutations can potentially alter the stability of the RdRp protein. Therefore, we propose that RdRp mutations in Indian SARS-CoV-2 isolates might have functional consequences that can interfere with RdRp targeting pharmacological agents.

Keywords: COVID-19; Indian geographical area; Mutation; Nsp12; RNA-dependent RNA polymerases (RdRp); SARS-CoV-2.

Figure 1. Multiple sequence alignment of SARS-CoV-2 RdRp protein.

Multiple sequence alignment of the Wuhan SARS-CoV-2 RdRp protein with sequences obtained from India. The mutations are highlighted in bold/italic font. Only those sequences are shown that have variations; the rest of the sequences are identical among all samples.

Figure 2. Prediction of secondary structure of RdRp protein.

Effect of mutations on secondary structure of RdRp. (A–H) demonstrate seven mutations observed in Indian isolates; (i) represents sequence of Wuhan isolate and (ii) represents sequence of Indian isolates. The small rectangular box shows the mutant residue. The difference of secondary structure between Wuhan and Indian isolates are highlighted with position of dashed box in respective panels.

Figure 3. Effect of mutations on structural dynamics of RdRp protein.

Analysis of RdRp dynamicity and flexibility. (A) The table shows the values of change in ΔΔS ENCoM and ΔΔG due to the mutation. (B, C and D) Δ Vibrational Entropy Energy between Wild-Type and Mutant RdRp, amino acids are coloured according to the vibrational entropy change as a consequence of mutation of RdRp protein. Blue represents a rigidification of the structure and red a gain in flexibility.

Figure 4. Effect of amino acid substitution on interatomic interactions.

Interatomic interactions mediated by A185V, I201L and P323L of RdRp- (A and B) represent alanine to valine substitution at 185^th position, (C and D) represent isoleucine to leucine substitution at 201^st position, (E and F) represent proline to leucine substitutions at 323^rd position. Wild-type and mutant residues are coloured inlight-greenand are also represented as sticks alongside with the surrounding residues which are involved on any type of interactions.