Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

Abstract

Background: Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood.

Methods: Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs.

Results: The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs.

Discussion: Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

Keywords: Capsid assembly; Circular dichroism; Fluorescence spectroscopy; HCV core protein; Structural biology.

Figure 1. HCV core protein.

(A) Primary sequence of HCV core protein. Hydrophobic residues (L, I, V, F, W and M) are shown in green, acidic residues in red and basic residues in blue. (B) Scheme of HCV core protein domains and their respective functions are highlighted.

Figure 2. Structural changes of HCV C124 induced by TFE and butanol.

Changes on the tertiary structure (A) and secondary structure (B) of C124 (25 µM) in the presence of 30% butanol or 30% TFE. Inset shows the resulting CD spectrum obtained after subtracting the C124 CD spectrum in the presence of TFE from that in the absence of TFE.

Figure 3. Analysis of binding of bis-ANS to C124.

(A) Analysis of bis-ANS binding to C124 (1 µM) by the increase of fluorescence intensity of bis-ANS spectra. (B) Changes on the secondary structure observed by Far-UV CD of C124 (25 µM) in the presence of the probe (25 µM). Inset: intrinsic fluorescence measurements of C124 in the absence and in the presence of bis-ANS at room temperature. (C) Double wavelength plot, [θ]₂₂₂ versus [θ]₂₀₀, modified from Uversky (2002), of a set of well-characterized unfolded, RC-like (dark green diamonds) or PMG-like proteins (dark red circles), and of the C124 in the absence or presence of Bis-ANS (25 µM) that the positions are highlighted (red and blue squares, respectively). (D) Plot of the ratio between the ellipticity at 222 nm and the ellipticity at 200 nm ([θ]₂₂₂/[θ]₂₀₀) of the same set of well-characterized RC-like or PMG-like proteins shown in (C). The position in the plot of C124 in the absence or presence of bis-ANS is highlighted (red and blue squares, respectively). The arrows in (C) and (D) are indicating the changes in the C124 position promoted by bis-ANS binding.

Figure 4. Effects of pH on the structure of C124.

(A) Changes in the intrinsic fluorescence induced by high pH values. (B) Light scattering of C124 at different pH values. (C) Temporal evolution analysis of in vitro assembly of NLPs at different concentrations of C124 triggered by the pH 12. (D) Plot of the maximum O.D. values at 350 nm as derived from the curves in (C) at different C124 concentrations.

Figure 5. Electron micrographs of negatively stained nucleocapsid-like particles (NLPs) produced from truncated HCV core protein at pH 12.

The protein concentration was 20 µM. Bars: 100 nm (A) and 300 nm (B and C).

Figure 6. Interaction of HCV core protein with unspecific nucleic acids and NLPs formation.

(A) Temporal evolution studies of in vitro assembly of NLPs at different concentrations of C124 triggered by the addition of 5 µM DNA poly(GC). (B) Heat flux profile associated with injections of 5 µM nucleic acid (poly(GC) DNA or RNA (SAF93^43–59)) in the calorimetric cell containing C124 (20 µM) at 37 °C. (C–E) Electron micrograph of negatively stained nucleocapsid-like particles (NLPs) produced from C124 at 2.5 µM (C), 10 µM (D) and 50 µM (E). To each protein concentration was added 5 µM poly(GC) DNA. Bars: (C)—100 nm and (D, E)—300 nm.

Figure 7. C-terminal truncated HCV core protein and unspecific nucleic acids (DNA or RNA) interaction analyzed by fluorescence correlation spectroscopy.

Fluctuation of the fluorescence intensity of RNA labeled with Alexa-488 in the absence (A) or in the presence (B) of C-terminal truncated HCV core protein, and the normalized autocorrelation curves of free DNA or RNA at 100 nM and in the presence of 1 µM or 500 nM of C124 (C). The buffer used was 10 mM phosphate (pH 7.4) with 100 mM NaCl.

Figure 8. Free energy diagram representing the energy barrier between the disordered state of C124 to oligomeric state (empty capsid or nucleic acid loaded capsid).