Sequence analysis of Indian SARS-CoV-2 isolates shows a stronger interaction of mutant receptor-binding domain with ACE2

Abstract

Objective: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the whole world, including Odisha, a state in eastern India. Many people have migrated to the state from different countries as well as other states during this SARS-CoV-2 pandemic. The aim of this study was to analyse the receptor-binding domain (RBD) sequence of the spike protein from isolates collected from throat swab samples of COVID-19-positive patients and further to assess the RBD affinity for angiotensin-converting enzyme 2 (ACE2) of different species, including humans.

Methods: Whole-genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19-positive patients was performed by ARTIC amplicon-based sequencing. Sequence analysis and phylogenetic analysis were performed for the spike region and the RBD region of all isolates. The interaction between the RBD and ACE2 of five different species was also analysed.

Results: The spike region of 32 isolates showed one or multiple alterations in nucleotide bases in comparison with the Wuhan reference strain. One of the identified mutations, at position 1204 (Ref A, RMRC 22 C), in the RBD coding region of the spike protein showed stronger binding affinity for human ACE2. Furthermore, RBDs of all the Indian isolates showed binding affinity for ACE2 of different species.

Conclusion: As mutant RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The binding affinity of the RBDs for ACE2 of all five species studied suggests that the virus can infect a wide variety of animals, which could also act as natural reservoir for SARS-CoV-2.

Keywords: ACE2; Indian isolates; Receptor-binding domain; SARS-CoV-2; Spike.

Figure 1

(A) Multiple sequence alignment of receptor-binding domain (RBD) protein sequences of Regional Medical Research Centre (RMRC) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates. All the sequences were aligned with the reference strain, and the arrow indicates the alteration found in the RBD of the RMRC 22 isolate. (B) Phylogenetic analysis of RBD protein sequences of RMRC isolates using the neighbour-joining method. The bootstrap consensus tree inferred from 1000 replicates has been taken to represent the evolutionary history of the taxa analysed. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed by the Poisson correction method and are in the unit of the number of amino acid substitutions per site.

Figure 2

Three-dimensional structure analysis and protein–protein interaction of angiotensin-converting enzyme 2 (ACE2) of Manis javanica with wild-type and mutant receptor-binding domain (RBD): (A) predicted 3D structure of ACE2; (B) Ramachandran plot and Z score; (C) ACE2 interaction with wild-type RBD; (D) ACE2 interaction with mutant RBD.

Figure 3

Three-dimensional structure analysis and protein–protein interaction of angiotensin-converting enzyme 2 (ACE2) of Mesocricetus auratus with wild-type and mutant receptor-binding domain (RBD): (A) predicted 3D structure of ACE2; (B) Ramachandran plot and Zscore; (C) ACE2 interaction with wild-type RBD; (D) ACE2 interaction with mutant RBD.

Figure 4

Three-dimensional structure analysis and protein–protein interaction of angiotensin-converting enzyme 2 (ACE2) of Rhinolophus sinicus with wild-type and mutant receptor-binding domain (RBD): (A) predicted 3D structure of ACE2; (B) Ramachandran plot and Zscore; (C) ACE2 interaction with wild-type RBD: (D) ACE2 interaction with mutant RBD.

Figure 5

Three-dimensional structure analysis and protein–protein interaction of angiotensin-converting enzyme 2 (ACE2) of Cynopterus sphinx with wild-type and mutant receptor-binding domain (RBD): (A) predicted 3D structure of ACE2; (B) Ramachandran plot and Zscore; (C) ACE2 interaction with wild-type RBD; (D) ACE2 interaction with mutant RBD.

Figure 6

Three-dimensional structure and protein–protein interaction of angiotensin-converting enzyme 2 (ACE2) receptor of Homo sapiens with wild-type and mutant receptor-binding domain (RBD): (A) 3D structure of ACE2; (B) predicted structure of wild-type and mutant RBD of Regional Medical Research Centre (RMRC) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates; (C) ACE2 interaction with wild-type RBD; (D) ACE2 interaction with mutant RBD.