Glycosylation studies of G-protein of BA genotype of group B human respiratory syncytial virus in mammalian cells

Human respiratory syncytial virus (hRSV) is the most common viral agent of Acute Respiratory infection (ARI). hRSV. The group B genotype (BA viruses) with 60 bp duplication in the second hypervariable region of G gene is the emerging hRSV genotype reported from India. The G glycoprotein (∼90 KDa) is extensively modified by addition of N and O-linked oligosaccharides. The present study is focused on the glycosylation of BA laboratory strain (281) in comparison to prototype hRSV (18537). The full length sequence of prototype group B strain and BA laboratory isolate was optimized and synthesized by commercial means and inserted in pUC 57 vector. The ectodomain G protein gene of hRSV with restriction site was amplified using synthetic sequence. The amplified fragments were cloned in pcDNA 3.1 eukaryotic expression vector with taq. The protein was expressed in Hep-2 cell line and purified by Ni-NTA affinity column. We used enzyme (Tunicamycin and Glycosidase F) based deglycosylation of the expressed protein to further investigate the N- and O- linked glycosylation of ectodomain G protein of hRSV. Our result indicates that the ectodomain G protein of hRSV has both N-linked and O-linked glycosylation with O-linked sugars contributing to the major part of the glycosylation. The BA viruses with 60 bp duplication showed enhanced glycosylation as compared to the prototype strain. It might be suggested that 60 bp duplication may have a role in immune evasion strategy of these current circulating strains of hRSV. Further studies with metabolic inhibitors of glycosylation process are needed to elucidate the detailed mechanism and sites of glycosylation of G protein of hRSV.