A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase

Abstract

Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Delta1 loop serves as a flexible locking mechanism to regulate the conformations needed for de novo initiation and for elongative RNA synthesis. A similar locking motif can be found in other viral RdRps. Products associated with the open conformation of the HCV RdRp were inhibited by interaction with the retinoblastoma protein but not cyclophilin A. Different conformations of the HCV RdRp can thus affect RNA synthesis and interaction with cellular proteins.

FIGURE 1.

Mutational analysis of the RNA channel in the HCV RdRp. A, a ribbon diagram representation of the structure of the HCV RdRp molecule(Protein Data Bank code 1QUV) and a cut away representation of the template channel. The thumb, finger, and palm subdomains are identified by the letters T, F, and P, respectively. The amino acids in the template channel mutated to alanine are shown with their side chains in different colors. The yellow residues are the aspartates that bind the divalent metals and are intended to serve as a landmark in the catalytic pocket. The blue residues identify substitutions that increased RNA synthesis. The red residues were inactive. The purple residues are defective in de novo initiation but competent for extension from a primed template. The mutations that did not significantly affect RNA synthesis (Table 1) are not shown in this figure. Distances shown were measured as follows: 18 Å is from OE1 of Gln⁴⁴⁶ to OE of Lys⁹⁸; 21.5 Å is from CB of Ser⁵⁵⁶ to OD1 of Asp³⁵²;4.5 Å is from NE2 of Gln⁴⁴⁶ to OE1 of Glu¹⁴³; 16.5 Å is from NE2 of Gln³⁵⁵ to CG of Lys¹⁵⁵.B, the sequence and predicted secondary structures of LE19, a template for RNA-dependent RNA synthesis. LE19 exists in two conformations, a stem-loop structure that can direct de novo initiation and a partially base-paired dimer that can direct primer extension. C, image of the products of RNA synthesis by the mutant RdRps. Mutant proteins tested in each reaction are shown at the top of the gel image. The purified proteins used in each reaction, resolved on 4-12% SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue are shown below the autoradiogram images. The protein molecular weight markers are the SeeBlue standards from Bio-Rad. The sizes of the RdRp products forde novo initiation (19 nt), PE (32 nt), and the purified HCV RdRps (65 kDa) are shown to the left of the gel images.

FIGURE 2.

His⁴²⁸ and Trp³⁹⁷ interact with the Δ1 loop. A, RNA products made by mutant and Δ21 using templates and reaction conditions to examine de novo initiation and PE. The proteins, templates, and the GTP concentrations used in the reactions are indicated above the gel image. LE19P is a version of LE19 that has a covalently attached 3′-puromycin. The lane numbers are indicated at the bottom of the gel image. The de novo initiated (19 nt) and primer extension products (32 nt) are indicated to the left of the gel image. B, RNA synthesis by the HCV RdRp with template derived from HCV. RNA H115 contains the 3′ 115 nt of HCV minus-strand RNA. RNA H115 + PE46 contains the PE46 sequence (boxed) fused to the 3′ end of H115. Whereas H115 can direct de novo initiation, H115 + PE46 is capable of primer extension. The gel images show the reaction products of the enzymes using H115 or H115 + PE46 as template, which were run on a 10% urea denaturing polyacrylamide gel. The divalent metal ion manganese was left out of the reaction mix in the assays with H115 and H115 + PE46 templates. The lane numbers are shown at the bottom, and the enzyme names are shown at the top of the lanes. The lengths of the products are shown to the left of the image; the 115 nt band corresponds to a de novo initiation product of H115, and the 275 nt band corresponds to the PE product of H115 + PE46. C, interaction between His⁴²⁸, Trp³⁹⁷, and the Δ1 loop. The Δ1 loop is rendered as a blue ribbon with the network of primarily hydrophobic interaction residues below, indicated in red, except for His⁴²⁸ and Trp³⁹⁷, which are in purple. The box contains a ribbon representation of the Δ1 loop (residues underlined) interacting with the helices in the thumb domain. The apex of the Δ1 loop is colored gold.

FIGURE 3.

Molecular modeling of the HCV RdRp, emphasizing the effect of the substitution on the Δ1 loop. A, location of Δ1 loop (colored in cyan) with respect to the thumb, palm, and fingers is shown in the crystal structure (1QUV) of the HCV NS5B protein. The Δ1 loop protrudes at the tip of the fingers domain and contacts the thumb domain by packing its short α-helix (helix A) against the α-helices O (residues 388-401) and Q (residues 418-437) of the thumb domain. The Trp³⁹⁷ residue is shown in purple. The active site is indicated by green stars. B, superposition of the Trp³⁹⁷ mutant model (red and sky blue) of NS5B and the WT structure (yellow and cyan). Only Δ1 loop and its contact regions are shown. The side chain of Trp³⁹⁷ stacks into the Δ1 loop. In the W397A mutant model, the key interactions are lost, and the Δ1 loop moves significantly away from its contact region. The red arrow indicates the movement. C, superposing a mutant in helix A of the Δ1 loop (L26A/S27A/N28A/S29A/L30A) and the wild-type protein; the Δ1 loop moves away from the thumb domain, and the helix A widens into a loop. The arrow indicates the direction of the movement.

FIGURE 4.

Biophysical and biochemical analyses of the mutant RdRps. A, analytical ultracentrifugation to examine the oligomerization state of W397A and Δ21 at two temperatures. The profiles for the two proteins largely overlap, suggesting that both are homogeneous and monomeric in solution. B, intrinsic fluorescence of the Δ21, W397A, H428A, and m26-30. The W397A has a spectrum indistinguishable from Δ21. H428A and m26-30 show a pronounced red shift in the spectrum between 349 and 363 nm. C, partial rescue of de novo initiation by dinucleotide primers. Shown is an autoradiograph of the RdRp products from template LE19 in the presence or absence of GTP or the NTPi mimic, GpU, as indicated above the gel image. D, PE from template PE46 in the presence of heparin. E, double-stranded RNA binding by Δ21 and the mutant RdRps. The gel image is from a 10% nondenaturing gel. The sequence of the probe used, RIII46, is shown above the gel image.

FIGURE 5.

Effects of recombinant cyclophilin A and pRb on RNA synthesis by the HCV RdRp. A, model of the HCV RdRp with the locations of residues that affect the activity of CypA or pRb in HCV-infected cells. The locations of Trp³⁹⁷ and His⁴²⁸ are in purple, and the catalytic aspartates responsible for coordinating divalent metals are in yellow. Ile⁴³², which has been identified to confer resistance to cyclosporin (27), the small peptide inhibitor of CypA, is in orange. Cys³¹⁶, which probably contacts pRb (23), is in light green. B, CypA can inhibit the HCV RdRp products made by de novo initiation and PE. The molar ratios of CypA or CypB to RdRp used in the reactions are listed above the gel image. C, quantification of the effects of CypB on de novo initiated and PE by Δ21. D, pRb can preferentially inhibit primer extension by Δ21. The gel image shows qualitatively the production of the de novo and primer extension products. The graph is from a related experiment, where pRb was titrated in increasing concentrations. All products made in three independent samples in the absence of pRb were set as 100%. E, effect of pRb on Δ21 and the mutant C316A, a part of the pRb binding site in NS5B (23). Where present, pRb was at a molar ratio of 22.5:1 to the RdRps. F, effects of pRb on RNA synthesis by the RdRps from JFH-1 2a HCV and BVDV. A 22.5:1 molar ratio of pRb to RdRp was used.

FIGURE 6.

Δ1 looplike structures in other viral RdRps. The space-filling models show the locations of the loops that could have a role comparable with that of the HCV Δ1 loop in the RdRp structures of HCV, Dengue virus, West Nile Virus (WNV), and BVDV. The schematic to the left shows the potential interactions that allow the comparable loops to interact with the outer surface of the template channels in the RdRps. Residues in the apex of the loop are in boldface type, and residues in the D2 loop are underlined. The key in the box shows the lines used for the different interactions.

FIGURE 7.

A model for RNA synthesis by the HCV NS5B. A schematic representation of the NS5B in the closed conformation is shown to the left with the thumb (T), fingers (F), and palm (P) domains holding a single-stranded RNA (red). The active site residues are shown as red asterisks. During elongation, the enzyme undergoes large conformational changes, including the displacement of the Δ1 loop to hold the growing nascent RNA-template hybrid (right).