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. 2012 Aug;86(15):7818-28.
doi: 10.1128/JVI.00457-12. Epub 2012 May 16.

Structural Analysis of Hepatitis C Virus core-E1 Signal Peptide and Requirements for Cleavage of the Genotype 3a Signal Sequence by Signal Peptide Peptidase

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Structural Analysis of Hepatitis C Virus core-E1 Signal Peptide and Requirements for Cleavage of the Genotype 3a Signal Sequence by Signal Peptide Peptidase

Verena Oehler et al. J Virol. .
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Abstract

The maturation of the hepatitis C virus (HCV) core protein requires proteolytic processing by two host proteases: signal peptidase (SP) and the intramembrane-cleaving protease signal peptide peptidase (SPP). Previous work on HCV genotype 1a (GT1a) and GT2a has identified crucial residues required for efficient signal peptide processing by SPP, which in turn has an effect on the production of infectious virus particles. Here we demonstrate that the JFH1 GT2a core-E1 signal peptide can be adapted to the GT3a sequence without affecting the production of infectious HCV. Through mutagenesis studies, we identified crucial residues required for core-E1 signal peptide processing, including a GT3a sequence-specific histidine (His) at position 187. In addition, the stable knockdown of intracellular SPP levels in HuH-7 cells significantly affects HCV virus titers, further demonstrating the requirement for SPP for the maturation of core and the production of infectious HCV particles. Finally, our nuclear magnetic resonance (NMR) structural analysis of a synthetic HCV JFH1 GT2a core-E1 signal peptide provides an essential structural template for a further understanding of core processing as well as the first model for an SPP substrate within its membrane environment. Our findings give deeper insights into the mechanisms of intramembrane-cleaving proteases and the impact on viral infections.

Figures

Fig 1
Fig 1
Effect of mutations in the GT3a core-E1 signal peptide on SPP proteolysis. (A) Amino acid sequences of the core-E1 signal peptide region for HCV genotypes 1 to 7. Representative strains for each genotype are shown in parentheses. The predicted n, h, and c regions within the signal peptide are indicated. The amino acid hydropathy at each position is indicated as neutral (n), hydrophobic (o), hydrophilic (i), or variable (v). (B) wt and mutant sequences for the core-E1 signal peptide from HCV3a-Gla examined with the SFV system. (C) HuH-7 cells were electroporated with in vitro-transcribed RNA from the indicated SFV constructs. Cell extracts were prepared at 16 h after electroporation and analyzed by Western blotting with an antibody against core. Open and closed circles indicate SP- and SPP-cleaved core species, respectively.
Fig 2
Fig 2
Adaptation of the JFH1 core-E1 signal peptide to encode wt and mutant GT3a sequences. (A) Sequences encoded in the core-E1 signal peptide region for wt JFH1 and wt and mutant GT3a constructs. (B) HuH-7 cells were electroporated with in vitro-transcribed RNA, and cell supernatants were harvested at 24-h intervals. Virus titers were determined by measurements of TCID50 values. Data shown represent the average values from three independent experiments for the following constructs: JFH1-GT3a-wt1 (□), JFH1-GT3a-wt2 (◆), and JFH1 (×). Error bars indicate standard deviations. (C) HuH-7 cells were electroporated with in vitro-transcribed RNA from the indicated constructs. Cell extracts were prepared at the times indicated after electroporation and analyzed by Western blotting with an anti-core antibody.
Fig 3
Fig 3
Virus production in HuH-7-derived shRNA cell lines. (A) HuH-7 cells expressing either a scrambled (sh-Scram-HuH-7) or SPP (sh-SPP-HuH-7) short hairpin RNA were analyzed by Western blotting with antibodies to SPP and actin. (B) Cells were electroporated, and cell supernatants were harvested at 24-h intervals. Virus titers were determined by measurements of TCID50 values; values represent the averages of data from three independent experiments. Data are shown as follows: ■, JFH1-GT3a-wt1 in sh-Scram-HuH-7 cells; ●, JFH1 in sh-Scram-HuH-7 cells; □, JFH1-GT3a-wt1 in sh-SPP-HuH-7 cells; ○, JFH1 in sh-SPP-HuH-7 cells.
Fig 4
Fig 4
Effects of mutations in the core-E1 signal peptide from GT3a on virus production and proteolysis of core. (A) HuH-7 cells were electroporated with in vitro-transcribed RNA, and cell supernatants were harvested at 24-h intervals. Virus titers were determined by measurements of TCID50 values. Data shown represent the average values from three independent experiments and are for the following constructs: JFH1-GT3a-wt1 (□), JFH1 (■), JFH1-GT3a-mut1 (○), and JFH1-GT3a-mut2 (●). Error bars indicate standard deviations. (B and C) HuH-7 cells were electroporated with in vitro-transcribed RNAs as indicated. (B) Cell extracts were harvested at 72 h and analyzed by Western blotting with antibodies against core and NS5A. (C) Cells were prepared for immunofluorescence at 72 h after electroporation and analyzed by confocal microscopy with antibodies against core and ADRP. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the scale bar represents 10 μm.
Fig 5
Fig 5
Structural analyses of the sp-E1 synthetic peptide. (A) Far-UV circular dichroism (CD) analysis of sp-E1 in various environments (the sp-E1 amino acid sequence is shown at the top of panel B). CD spectra were recorded in water (H2O), complemented with either 50% 2,2,2-trifluoroethanol (TFE), 1% l-α-lysophosphatidyl choline (LPC), or the following detergents: 100 mM sodium dodecyl sulfate (SDS), 100 mM N-dodecyl-β-d-maltoside (DM), or 100 mM dodecyl phosphocholine (DPC). (B to D) NMR analysis of the sp-E1 peptide in 50% TFE. The two lysine residues in blue at the N terminus were added to increase the solubility of the peptide. The four C-terminal residues in magenta correspond to the first residues of the E1 glycoprotein. (B) Summary of sequential (i, i + 1) and medium-range (i, i + 2 to i, i + 4) NOEs. Sequential NOEs allowing the assignment of proline residues are indicated in red. Asterisks indicate that the presence of an NOE cross peak was not confirmed because of overlapping resonances. Intensities of NOEs are indicated by the heights of the bars. (C and D) NMR-derived 1H-α (C) and 13C-α (D) chemical shift differences were calculated by the subtraction of the experimental values from the reported random-coil-conformation values in TFE (28). The dashed lines indicate the standard threshold value of ΔH-α (−0.1 ppm) (C) or ΔC-α (0.7 ppm) (D) for an α-helix. (E) Amino acid sequence of the core-E1 signal peptide from strain JFH1 (amino acid residues 171 to 191), including the first four residues of the E1 glycoprotein (shown in magenta at the C terminus). The box indicates α-helix residues 175 to 185, revealed by NMR. Residues are color coded according to their physicochemical properties: hydrophobic residues are black, neutral residues (Ala and Gly) are gray, polar residues (Ser and Thr) are yellow, and Cys is green. The characteristic structural topology of signal peptide domains is indicated at the top, including the N-terminal domain (n), the hydrophobic core region (h), and the C-terminal domain (c) (53) (see the text for details). (F) Superimposition of the backbone heavy atoms (N, C′, and C-α) of the 27 final structures (PDB accession number 2kqi), calculated by using the standard simulated annealing protocol in the Xplor-NIH 2.2.1 program (44). The 27 structures were superimposed for the best overlap of residues 175 to 185. Average positions of side-chain residues reported as potential C-terminal residues of the mature core protein (see the text) are displayed (stick representation). (G) Ribbon and stick models of a representative experimental structure of the sp-E1 peptide. Residues are colored based on the chemical properties of their side chains, as indicated above. (H) Two views of the surface of α-helix residues 175 to 185 showing the relatively polar face versus the highly hydrophobic side rotated by 180° (top and bottom views, respectively). (I) Tentative position of the sp-E1 peptide within a phospholipid bilayer of POPC (1-palmitoyl-2-oleoyl-3-sn-glycero-3-phosphocholine) on the same scale. Figures were generated from structure coordinates using VMD (http://www.ks.uiuc.edu/Research/vmd/) (15) and rendered with POV-Ray (http://www.povray.org/).

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