A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism

Abstract

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process

“…Therefore, HCV fusion is most likely mediated by both E1 and E2, depending on intra-and intermolecular interactions to drive conformational rearrangements of the heterodimer required for fusion. Consistent with this model, interactions between E1 and E2 are critical for entry [68,111] and recent coevolution analysis revealed that E1 and E2 refold interdependently during fusion [112]. In a chaperone-like role, E2 likely supports the fusion properties of E1.…”

Flexibility and intrinsic disorder are conserved features of hepatitis C virus E2 glycoprotein

“…It is, therefore, very likely that certain regions of E2 are buried in the heterodimer and/or oligomer. Although there is no molecular structure for the E1E2 complex, various mutagenesis and evolutionary analyses has provided consistent reports on the E1-E2 interface, which is likely to include elements of HVR-2, the backlayer and the E2 stalk (the latter is not included in the ectodomain models) [9,[72][73][74][75]. Therefore, although HVR-2 exhibits high flexibility in our simulations, it is likely to be constrained within the E1E2 complex.…”

Disease-relevant mutations alter amino acid co-evolution networks in the second nucleotide binding domain of CFTR

“…The N1303T and D1270N MSAs had reduced number of sequences, 87 and 23 respectively, and these small datasets are not optimal for use with ELSC, OMES, and McBASC analyses. However, Blocks In Sequences (BIS) analysis is specifically optimized to detect coevolution patterns for MSAs with reduced numbers of sequences (around 50 or less) that have low evolutionary divergence that is typically found in vertebrate and viral protein families [39,44,54]. BIS analysis is a combinatorial method that provides a coevolution score for pairs of positions in an MSA and clusters positions with similar coevolution scores.…”