Near-Neighbor Interactions in the NS3-4A Protease of HCV Impact Replicative Fitness of Drug-Resistant Viral Variants

Abstract

A variety of amino acid substitutions in the NS3-4A protease of the hepatitis C virus lead to protease inhibitor (PI) resistance. Many of these significantly impair the replication fitness of the resistant variants in a genotype- and subtype-dependent manner, a critical factor in determining the probability with which resistant variants will persist. However, the underlying molecular mechanisms are unknown. Here, we present a novel residue-interaction network approach to determine how near-neighbor interactions of PI resistance mutations in NS3-4A can impact protease functional sites dependent on their genomic background. We constructed subtype-specific consensus residue networks for subtypes 1a and 1b from protease structure ensembles combined with biological properties of protein residues and evolutionary amino acid conservation. By applying local and global network topology analysis and visual exploration, we characterize PI resistance-associated sites and outline differences in near-neighbor interactions. We find local residue-interaction patterns and features at protease functional sites that are subtype specific. The noncovalent bonding patterns indicate higher fitness costs conferred by PI resistance mutations in a subtype 1b genomic background and explain the prevalence of Q80K and R155K in subtype 1a. Based on local residue interactions, we predict a subtype-specific role for the protease residue NS3-Q80 in molecular mechanisms related to the assembly of infectious virus particles that is supported by experimental data on the capacity of Q80K variants to replicate and produce infectious virus in subtype 1a and 1b cell culture.

Keywords: hepatitis C virus; molecular determinants; residue networks; subtype; viral variant fitness.

Fig 1.. 3D protein structure of the NS3–4A protease domain.

The protein structure shows a subtype 1a NS3 protease domain from Protein Data Bank entry 2OC8 co-crystallized with NS4A (light blue) and a linear protease inhibitor (PI) (purple stick model). The protein backbone is given as ribbon model with transparent surface depiction. Residues where PI resistance-associated amino acid substitutions occur are highlighted as orange stick models; functionally important residues are shown as green stick models. Functionally important sites are indicated by arrows: allosteric site (Allo); protease active/catalytic site (Cat); domain-domain interaction sites (DDIP); linker region (Linker); MAVS binding site (MAVS); binding site for the natural protease substrate (NPS); SOS-binding site for TRIF (SOS).

Fig 2.. Network comparison view between subtype 1a and 1b consensus residue-interaction networks with focus on functional sites and protease inhibitor resistance-associated sites.

This comparison network view shows the noncovalent residue interactions between a subset of residues of the NS3–4A protease with focus on the differences between subtype 1a and 1b consensus residue-interaction networks. The view focuses on the protease inhibitor (PI) resistance-associated sites 36, 54, 55, 80, 155, 156, 168, and 170 and further includes their direct neighbors, all residues annotated as functionally important (see Materials and Methods), and residues that vary between subtype 1a and 1b. Functional residue annotations are shown as vertical bars (cyan – allosteric site; red - catalytic site; green - domain-domain interaction sites; violet - residues interacting with the linker region; orange - MAVS peptide; blue - natural peptide substrate; yellow - linear PI; purple - SOS-binding site of TRIF). The residue frequency distance is mapped to the node color using a white-to-grey gradient for small-to-large values. Solid edge lines represent noncovalent residue interactions present in both subtype consensus residue-interaction networks, dashed edge lines are residue interactions from the subtype 1a consensus residue-interaction network, and dotted edge lines correspond to residue interactions in the subtype 1b consensus residue-interaction network. Edges that represent contacts are colored in blue, hydrogen bonds in red, and overlaps in grey; a darker color indicates interaction between main chain atoms, while lighter color as well as an arrow stands for side chain atom interactions.

Fig 3.. Network comparison view and corresponding 3D structure close-up for the subtype-specific protease inhibitor resistance-associated sites: (A) 36, (B) 54, (C) 80, (D) 155, (E) 156, and (F) 168.

Each comparison network view shows the noncovalent residue interactions between the specified residue and its direct neighbors from the subtype 1a and 1b consensus residue-interaction networks. Node and edge colors as well as functional annotations are described in the graphical legend above and in the Fig 2 legend. The corresponding residues from the subtype 1a reference structure from Protein Data Bank entry 2OBQ and the subtype 1b reference structure from Protein Data Bank entry 1DXP are aligned and shown as blue and orange sticks, respectively.

Fig 4.. Network comparison view between wild-type and mutant structure for V36M and R155K and corresponding 3D structure close-up.

(A) The comparison network view between the subtype 1a consensus residue-interaction network and the residue-interaction network of the V36M mutant structure from Protein Data Bank entry 2QV1 focuses on the direct noncovalent interactions of residue 36 with its neighbors. The corresponding residues in the structure are shown as blue and orange sticks, respectively. (B) The comparison network view between the subtype 1a consensus residue-interaction network and the residue-interaction network of the R155K mutant structure from Protein Data Bank entry 2OIN focuses on the direct noncovalent interactions of residue 155 with its neighbors. The corresponding residues in the structure are shown as blue and orange sticks, respectively.

Fig 5.. Comparison of replication capacity and infectious virus yield of H77S.3 versus N.2 RNAs with the Q80K substitution in the NS3–4A protease.

(A) The effect of NS3-Q80K on the RNA replication capacity was measured by GLuc assay in the H77S.3/GLuc2A (left, upper panel) or the N.2/GLuc2A genetic background (left, lower panel). Results are normalized to GLuc activity at 6 hrs post transfection, and represent the mean ± standard deviation of triplicate samples. (B) The effect of NS3-Q80K on the infectious virus production from the H77S.3 (right, upper panel) or the N.2 background (right, lower panel) was measured by a focus forming unit (FFU) assay from cell culture supernatant fluids collected every 24 hr post transfection. Data represent the mean ± standard deviation from at least 3 independent experiments.