A holistic evolutionary and structural study of flaviviridae provides insights into the function and inhibition of HCV helicase

Abstract

Viral RNA helicases are involved in duplex unwinding during the RNA replication of the virus. It is suggested that these helicases represent very promising antiviral targets. Viruses of the flaviviridae family are the causative agents of many common and devastating diseases, including hepatitis, yellow fever and dengue fever. As there is currently no available anti-Flaviviridae therapy, there is urgent need for the development of efficient anti-viral pharmaceutical strategies. Herein, we report the complete phylogenetic analysis across flaviviridae alongside a more in-depth evolutionary study that revealed a series of conserved and invariant amino acids that are predicted to be key to the function of the helicase. Structural molecular modelling analysis revealed the strategic significance of these residues based on their relative positioning on the 3D structures of the helicase enzymes, which may be used as pharmacological targets. We previously reported a novel series of highly potent HCV helicase inhibitors, and we now re-assess their antiviral potential using the 3D structural model of the invariant helicase residues. It was found that the most active compound of the series, compound C4, exhibited an IC50 in the submicromolar range, whereas its stereoisomer (compound C12) was completely inactive. Useful insights were obtained from molecular modelling and conformational search studies via molecular dynamics simulations. C12 tends to bend and lock in an almost "U" shape conformation, failing to establish vital interactions with the active site of HCV. On the contrary, C4 spends most of its conformational time in a straight, more rigid formation that allows it to successfully block the passage of the oligonucleotide in the ssRNA channel of the HCV helicase. This study paves the way and provides the necessary framework for the in-depth analysis required to enable the future design of new and potent anti-viral agents.

Keywords: Antiviral bioinformatics; Hepatitis C; Molecular dynamics; Structure-based drug design.

Figure 1. Phylogenetic reconstruction of flaviviridae NS3 protein sequences.

The tree shown is the best Bayesian topology. Numerical values at the nodes of the tree (x/y/z) indicate statistical support by MrBayes, PhyML and RAxML (posterior probability, bootstrap and bootstrap, respectively). Values for highly supported nodes have been replaced by symbols, as indicated. Full details and accession numbers for all protein sequences used are given in Table S1. The tree confidently separates the Hepacivirs, Pestivirus, and Flavivirus genera. Within the Flavivirus, TABV and insect-specific species appear basal, whereas Tick-borne species and species with no known vector (NKV) are the most derived. The inset shows the same tree in unrooted star format to better illustrate the relationships and distances between the subgroups.

Figure 2. Phylogenetic reconstruction of Hepacivirus NS3 protein sequences.

The tree shown is the best Bayesian topology. Numerical values at the nodes of the tree (x/y/z) indicate statistical support by MrBayes, PhyML and RAxML (posterior probability, bootstrap and bootstrap, respectively). Values for highly supported nodes have been replaced by symbols, as indicated. Full details and accession numbers for all protein sequences used are given in Table S1. The tree indicates two subgroups, one consisting of GBVA, GBVB, GBVC, and GBVD, and the other consisting of CHV and the six HCV genotypes. The inset shows the same tree in unrooted star format to better illustrate the relationship and distance between the subgroups.

Figure 3. Conserved residues identified by analysis of all flaviviridae NS3 proteins.

Conservation, quality and consensus tracks from Jalview for selected regions of the alignment shown in Fig. S2 are shown. Regions of high conservation are underlined with black boxes. The conservation annotation histogram (top) reflects conservation of the physicochemical properties, and marks absolutely conserved residues (score 11) with a yellow asterisk ‘*’, and columns where physicochemical properties are conserved (score 10) with a yellow ‘+’; less conserved positions are shown in darker colours with decreasing score. The quality annotation histogram (middle) reflects the likelihood of observing a mutation in any particular column of the alignment based on the BLOSUM62 matrix scores (for each column, the sum of the ratios of the two BLOSUM62 scores for a mutation pair, and each residue’s conserved BLOSUM62 score, are normalised and plotted on a scale of 0 to 1). The consensus histogram (bottom) reflects the percentage of the modal residue per column, and the consensus sequence logo is shown for conserved regions (‘+’ denotes non-conserved residues and ‘-’ denotes gap residues).

Figure 4. The 3D structure of HCV helicase showing all conserved amino acid groups highlighted in Fig. 3 in all four helicase X-ray structures.

The two arginine residues are indicated by black arrows. The route that the ssRNA fragment follows through the helicase is shown by the red arrow. A, B and C are the three regions of sequence and structural conservation. Four X-ray determined helicase structures have been superposed here. HCV helicase is orange, Dengue helicase is magenta, Yellow fever helicase is pink and Murray Valley Encephalitis helicase is turquoise.

Figure 5. Stochastic conformational search for C12 and C4.

It was established that C12 only formed two relatively close clusters (coloured red), whereas C4 formed two sets of conformationally distant ones (coloured green).

Figure 6. Conformational analysis for compounds C4 and C12.

(A) The most prominent conformations adopted by C4 and C12. (B) C4 in the ssRNA (blue ribbon) of HCV helicase. (C, Upper) The interaction of C4 with both arginine residues of the ssRNA channel is blocking the passage of the oligonucleotide. (C, Lower) Having established the Cys431 bond, C12 fails to reach Arg393 and form a blocking bridge in the ssRNA channel of the HCV helicase. Compounds have been color-coded as in A. Electrostatic potential surfaces for C4 have been drawn in B and C.