Coordinated evolution of the hepatitis C virus

Abstract

Hepatitis C virus is a genetically heterogeneous RNA virus that is a major cause of liver disease worldwide. Here, we show that, despite its extensive heterogeneity, the evolution of hepatitis C virus is primarily shaped by negative selection and that numerous coordinated substitutions in the polyprotein can be organized into a scale-free network whose degree of connections between sites follows a power-law distribution. This network shares all major properties with many complex biological and technological networks. The topological structure and hierarchical organization of this network suggest that a small number of amino acid sites exert extensive impact on hepatitis C virus evolution. Nonstructural proteins are enriched for negatively selected sites of high centrality, whereas structural proteins are enriched for positively selected sites located in the periphery of the network. The complex network of coordinated substitutions is an emergent property of genetic systems with implications for evolution, vaccine research, and drug development. In addition to such properties as polymorphism or strength of selection, the epistatic connectivity mapped in the network is important for typing individual sites, proteins, or entire genetic systems. The network topology may help devise molecular intervention strategies for disrupting viral functions or impeding compensatory changes for vaccine escape or drug resistance mutations. Also, it may be used to find new therapeutic targets, as suggested in this study for the NS4A protein, which plays an important role in the network.

Fig. 1.

Descriptive measures of the HCV network structure. (A) Degree distribution of the HCV network (solid line) and expected power law distribution (dashed line). (B) Clique distribution by size in the HCV network (black bars) and in 10,000 randomly rewired graphs (gray bars) that keep constant the number of vertices and their degree (19). (C) Rc of the HCV network (solid line) and the average of 10,000 randomly rewired graphs (dashed line) (20). (D) Structural robustness of the HCV network. The x axis is the percentage of removed nodes, and the y axis is the percentage of the original global efficiency of the network (21). The dashed line shows the random removal of nodes (average of 100 random sequences of node removal). The solid line shows the removal of nodes with the highest betweenness centrality (22). After each node removal, the betweenness centrality of each node was recalculated.

Fig. 2.

The HCV network. A genomic network of HCV coordinated substitutions is shown, where a vertex is an amino acid site and a link between two vertices is a significant physicochemical correlation between two sites. The position of each vertex depends on its k-shell value and on the k-shell value of its neighbors. A color code allows for the identification of k-shell values, and the vertex's size is proportional to its degree. The k-shell decomposition and visualization was performed with LaNet-vi (31).

Fig. 3.

HCV network and natural selection. (A) Sites that compose the HCV network and their k-shell value, which is shown as a moving average (window size = 9; step = 1). (B) Average values of k-shell for each protein (squares), calculated by using 404 sites in the network, and the average of the dS/dN ratio (circles) calculated over all of the sites of the protein. The correlation between the average k-shell and dS/dN values in HCV proteins is 0.5930 (P = 0.00001). (C) Scatter plot of HCV proteins. The x axis is the percentage of the sites of each protein that are located in the nucleus (k-shell, 36) and y axis the average dS/dN.

Fig. 4.

Hypothetical molecular interactions between NS3, NS4A, and NS5A. The links between sites are shown as green lines (interprotein) or orange lines (intraprotein). Sites located in the nucleus of the HCV network are shown in red. NS3, 3D structure of the NS3 protease (sites 1029 to 1205). The first 28 N-terminal aa involved in the molecular interaction with its NS4A cofactor are shown in yellow and red. NS5A, 3D structure of NS5A (sites 2008 to 2170). The sites involved in the molecular interaction with NS4A (2135 to2139) are shown in yellow and red. NS4A, All 54 sites of NS4A are shown as a contiguous chain divided in four domains: the membrane anchor (green), the NS3 cofactor (yellow), the kink (blue), and the acidic domain (cyan).