Mutations prevalent in fusion protein signal peptides from African RSV strains

Abstract

Background: Two subgroups of RSV are recognized namely, RSV-A and RSV-B. There are two major glycoproteins found on the surface of the virion, F and G glycoprotein. The F protein which is the more conserved major glycoprotein is the main target for vaccine development. The F protein comprises of a signal peptide which is the N terminal extension that targets the F protein to the different secretary pathways in the cytoplasm. Mutations found in the signal peptide may affect the control of gene expression, replication and pathogenicity. This study aimed to investigate the diversity and impact of the most prevalent signal peptide mutations on the expression of F proteins from African RSV strains. Methods: 4876 African RSV genome and F gene sequences were retrieved from GenBank and GISAID. Nextclade was used to align F protein sequences and to construct phylogenetic trees. 3841 sequences were excluded due to lack of full complete RSV F genome, and the remaining 1035 were compared to the RSV-A A2 long strain and RSV-B B1 reference strains using BioEdit and AliView. These two software tools were also used to edit and trim the sequences and further assessed the positional amino acid frequency in signal peptides alignment. The identified mutations on the signal peptide were introduced into the RSV F protein sequence using site-directed mutagenesis and western blot was done to detect the expressed RSV F proteins. Results: Among 1035 African RSV F sequences from 2007-2022, the most prevalent subtype was RSV-B at 89,9% (930/1035). The dominant genotypes among RSV-A strains were GA2.3.5 prevalent at 47.6% (50/105) and GA2.3.3 was 34.3% (36/105). Both belong to clade A23. While for RSV-B, GB5.0.5a was prevalent at 66.9% (622/930) and GB5.0.2 at 28.9% (269/930) and belongs to clade B6. Mutations in RSV-A F signal peptides were identified at four amino acid positions (I5T, A8T, T12A/I and L15F) at frequencies of <3%. T12A/I and T8I/A mutations were found in genotype GA2.3.5 (clade A23) South African sequences from 2018 and 2020. The I5T and L15F mutations were among Egyptian strains from 2018-2019. L4P, H6Y, F12L and L15F mutations were observed in RSV-B strains. The RSV F protein was expressed in the HEK-293T cells. The soluble protein fractions contained proteins of 18kDa that corresponded to the expected size of the F2 subunit for mutants that contain the T12I substitution. The I5T mutant protein appeared as a band between 45-55kDa, corresponding to the size of the cleaved F1 subunit. However, subsequent sequencing revealed that none of the expressed proteins have the expected amino acid substitutions. This ultimately meant that our site-directed mutagenesis experiments to introduce the mutations of interest failed. Discussion: RSV-A mutations were identified at 7/15 amino acid positions and 4/7 positions (5, 8, 12, 15) were the most prevalent, and globally T12I and L15F were the most prevalent. In RSV-B, 7/15 mutations were identified on the signal peptide and 4/7 positions (4, 6, 12, 15) were the most prevalent. F12L and L4P were the most prevalent mutations globally. In general, we Page 6 of 91 observed that RSV-B signal peptide mutations occurred at a lower frequency. However, we were unable to recover F proteins with mutated signal peptides.