A computational approach identifies two regions of Hepatitis C Virus E1 protein as interacting domains involved in viral fusion process

Abstract

Background: The E1 protein of Hepatitis C Virus (HCV) can be dissected into two distinct hydrophobic regions: a central domain containing an hypothetical fusion peptide (FP), and a C-terminal domain (CT) comprising two segments, a pre-anchor and a trans-membrane (TM) region. In the currently accepted model of the viral fusion process, the FP and the TM regions are considered to be closely juxtaposed in the post-fusion structure and their physical interaction cannot be excluded. In the present study, we took advantage of the natural sequence variability present among HCV strains to test, by purely sequence-based computational tools, the hypothesis that in this virus the fusion process involves the physical interaction of the FP and CT regions of E1.

Results: Two computational approaches were applied. The first one is based on the co-evolution paradigm of interacting peptides and consequently on the correlation between the distance matrices generated by the sequence alignment method applied to FP and CT primary structures, respectively. In spite of the relatively low random genetic drift between genotypes, co-evolution analysis of sequences from five HCV genotypes revealed a greater correlation between the FP and CT domains than respect to a control HCV sequence from Core protein, so giving a clear, albeit still inconclusive, support to the physical interaction hypothesis.The second approach relies upon a non-linear signal analysis method widely used in protein science called Recurrence Quantification Analysis (RQA). This method allows for a direct comparison of domains for the presence of common hydrophobicity patterns, on which the physical interaction is based upon. RQA greatly strengthened the reliability of the hypothesis by the scoring of a lot of cross-recurrences between FP and CT peptides hydrophobicity patterning largely outnumbering chance expectations and pointing to putative interaction sites. Intriguingly, mutations in the CT region of E1, reducing the fusion process in vitro, strongly reduced the amount of cross-recurrence further supporting interaction between this region and FP.

Conclusion: Our results support a fusion model for HCV in which the FP and the C-terminal region of E1 are juxtaposed and interact in the post-fusion structure. These findings have general implications for viruses, as any visualization of the post-fusion FP-TM complex has been precluded by the impossibility to obtain crystallised viral fusion proteins containing the trans-membrane region. This limitation gives to sequence based modelling efforts a crucial role in the sketching of a molecular interpretation of the fusion process. Moreover, our data also have a more general relevance for cell biology as the mechanism of intracellular fusion showed remarkable similarities with viral fusion.

Figure 1

Schematic diagram of the E1 regions analyzed in this study. The Kyte & Doolittle (continuous line) and Goldman (dashed line) hydrophobicity plots of the E1 protein is shown at the top. The studied E1 fragments are indicated as shaded boxes. The trans-membrane (TM) domain is also shown. Key amino acid positions are indicated.

Figure 2

An example of cross-recurrence plot of the FP-CT comparison (genotype 1b). Each black dot represents a cross-recurrence, i.e. very similar hydrophobicity between the compared amino acid residues from FP and CT peptides.

Figure 3

DET and REC values from (A) autologous and heterologous couples and from (B) FP-CT and other autologous couples. (A) Autologous pairs (FP/IT, FP/CT, IT/CT) (black dots) are more correlated than heterologous ones (FP/Core, CT/Core, IT/Core) (white dots). (B) Differently from other autologous couples (white dots), FP/CT couples (black dots) seem to occupy (with one only exception) the most extreme right portion of the graph, so indicating a possible preferential FP-CT attachment pairs.