Investigating the Molecular Details of Hepatitis C Virus Entry Events

Abstract

The RNA genome containing Hepatitis C Virus (HCV), remains an enigma due to the presence of many genotypes and quasispecies. To understand genotypic differences of HCV glycoprotein E2, 29 nucleotide sequences of genotypes 1 and 3 were analysed by in silico studies. Nucleotide analysis revealed a preference for Cytosine which was attributed to a bias for Cytosine rich codon in genotype 1 and a similar but lower preference in genotype 3. Comparison of amino acids revealed a relatively conserved N-terminal region with majority of the inter-genotypic changes concentrated at the C-terminal portion. Modelling followed by docking analysis between de novo models of E2 glycoproteins and receptor protein CD81 revealed unique interacting residues for both proteins with genotype 3a exhibiting the strongest interaction with the receptor. In our quest to understand the early events of HCV entry, fluorescent pseudoparticles (HCVpp) were generated and used to study HCV trafficking. We observed internalization and recycling of fluorescence within 15 minutes of HCVpp entry and maximum colocalisation with plasma membrane marker FM 4-64 also at 15 minutes. We predict that fusion of viral and cellular membranes occurred within this time. To understand the role of regulatory GTPases Rab1a, similar experiments were repeated in a Rab1a knockdown cell lines. In these cells, we observed impaired trafficking indicating an important role of Rab1a in HCV entry. To identify entry inhibitors of HCVpp from medicinal plants, methanolic leaf extracts were prepared from Psidium guajava L., Plumeria alba L., Syzygium cumini L. and Tamarindus indica L., plants with antiviral and hepatoprotective property. We observed that Tamarindus extract exhibited an inhibitory role by both qRT-PCR and microscopic experiments. In conclusion, we have established stark differences in structure and binding activities of the glycoprotein E2 across genotypes 1 and 3. We have established a trafficking assay using fluorescent HCVpp and predict a probable time line of HCV entry. We have also reported a role of Rab1a in HCV trafficking. To establish a potent natural HCV therapeutics, we found Tamarind leaf extract to act as a potent entry inhibitor for the virus. However further mechanistic details still need to be investigated.