De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5

Abstract

Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V(max)/K(m) ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in V(max) values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.

Figure 1. Amino acid sequences of the isolates used in this study.

Alignment of the predicted amino acid sequences (Δ21) of the NS5B proteins used in this study and the consensus sequences corresponding to subtypes 1b, 2a, 3a and 5a. As only 1 sequence for subtype 4d was present in the http://hcv.lanl.gov/the consensus for NS5BΔ-4d corresponds to the whole genotype 4. Only differences with a general consensus sequence are shown. Gray boxes indicate the sequences corresponding to αhelixes C, F, K, L, and N.

Figure 2. Phylogenetic analysis.

Neighbor-joining phylogram of NS5BΔ21 from this study (G1, G2, G3, G4 and G5) and the same reference sequences as in A). A consensus for genotype 6 has also been included. Bootstrap analysis was performed with 100 repetitions. *  =  bootstrap values of 100% showing the divergence of the different genotypes.

Figure 3. Purification of NS5BΔ21 proteins by affinity chormatography and cationic exchange.

A) Purified proteins after cationic exchange chromatography (heparin-sepharose column) eluted at 500 mM NaCl and detected with Coomassie blue staining. B) Western blot detection of HCV NS5B genotypes 1 to 5 with a polyclonal antibody. Protein molecular weigth markers (in kilodaltons) are shown on the left.

Figure 4. Characterization of the conditions for optimal RNA synthesis in a “de novo” initiation process by polymerases from different genotypes.

The ability of each polymerase to synthesize poly-G from [α-³²P]GTP using poly-C template is normalized with respect to its maximum activity. The effect of the following conditions was analyzed: A) MnCl₂ concentration (metal ion requirements), B) pH and, C) NaCl concentration (ionic strength). Reaction mixtures contained 125 µM GTP and 40 ng/µl of polyC. Reactions in panel B) were performed with different buffers according to the selected pH: sodium acetate for pH 5, MES for pH 6, MOPS for pH 7.25, HEPES for pH 7.75, Tris-HCl for pH 8.25 and CAPS for pH 10. All graphs show means of at least three independent experiments. Error bars were lower than 20% in all points and have been removed for clarity.

Figure 5. NS5B oligomerization of genotypes 1 to 5.

A) Spectra obtained for a representative FRET experiment. Proteins NS5BΔ21 fused to cyan and NS5BΔ21 fused to citrine were mixed at eqimolar concentration in FRET buffer. Then, the mixture was excited at 432 nm and spectra were recorded from 460 nm to 600 nm. Two main peaks were obtained, one corresponding to cyan at approximately 478 nm and the other at approximately 530 nm corresponding to citrine. FRET ratios were calculated as the ratio of the 530 nm and 478 nm intensities. B) FRET ratios for the interaction of eqimolar amounts (50 nM each) of NS5BΔ21-cyan and NS5BΔ21-citrine for each genotype are shown. Spectra were obtained in the presence of 10 mM NaCl and 4.5 mM Mg(CH₃COO)₂. Values are normalized against the ratio obtained for NS5BΔ21-1b and expressed in percentage. Results are the mean and SEM of twelve independent experiments. C) FRET ratios were calculated as described above, unless spectra were obtained in the presence of 66 mM NaCl and 5 mM MnCl₂. Ratios are normalized against the ratio obtained for NS5BΔ21-1b, and expressed in percentages. Values are the mean and SEM of six independent experiments.

Figure 6. NS5B electrostatic surface potential.

A) Ribbon diagram within PYMOL 1.3 showing the overall structure of HCV NS5B based on the HC-J4 structure reported by Jäger and coworkers . The symmetrical locations of F and T helices as well as the primary sequence corresponding to αF helix for the genotypes used in this study are shown. B) Location of electrostatic surface potential for the putative ligand site in the region corresponding to αF helix (Ser112-Asp129) of NS5B. C) Location of the electrostatic surface potential for the putative receptor site in the region corresponding to αT (Pro495-Arg505. D) Ligand electrostatic surface potential for NS5B from genotypes G1b, G2a, G3a, G4d and G5a. In silico mutagenesis were performed using Foldx as described in Materials and Methods. The symbols > or  =  compare the intensity of electrostatic surface potential on each phenotype. For panels B, C and D, the color coded electrostatic surface potential was drawn using the Adaptative Poisson-Boltzmann equation as described in Materials and Methods .

Figure 7. NS5B/NS5B docking model complexes showing the sites of protein-protein interaction and the amino acids involved in ionic interactions.

The side chain of amino acids that form ion pairs is highlighted with red color for acidic amino acids and blue color for basic amino acids. Green and yellow colors for ribbons represent the putative ligand and receptor partners, respectively. Models 091, 004, 034, 042, 050, and 061 are represented in panels A, B, C, D, E, and F, respectively. These models were obtained as described in Materials and Methods, and Table 3.