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

Abstract

BackgroundHepatitis 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.MethodsSpectroscopic 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.ResultsThe 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.DiscussionTogether, 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.

The interaction of dengue virus capsid protein with negatively charged interfaces drives the in vitro assembly of nucleocapsid-like particles

“…We believe that this different result can be explained by the optimized conditions for sample preparations for AFM or TEM imaging, i.e., drying very diluted DENVC solutions over negatively charged surfaces (mica or grid carbon layer, respectively). Accordingly, self-assembly in the absence of nucleic acid has been also reported for HCV [ 43 , 44 ]. The unique features of flaviviruses’ C proteins, such as its small size and the highly positive surface electrostatic potential [ 22 ] (see Fig 5 ), can further facilitate interaction with the solid charged substrate, driving the self-assembly process.…”

Self-assembly of dengue virus empty capsid-like particles in solution

“… 29 Indeed, either for flaviviruses or for alphaviruses, which are also small enveloped +ssRNA viruses, attempts for reproducing NC assembly in vitro required capsid protein charge neutralization. 28 , 30 , 31 , 32 Thus, it is expected that genomic RNA elements, namely negative charges, and long length, drive the orientation of capsid proteins to allow the protein-protein interaction necessary to build the capsid. Our TEM results showed an average diameter size of ∼30 nm for the CLPs formed by the R85C ox mutant.…”

Elucidating the Thermodynamic Driving Forces of Polyanion-Templated Virus-like Particle Assembly

“…32,33 Moreover, ITC has been applied by De Souza et al to qualitatively study the assembly of hepatitis C virus core proteins around a nucleic acid. 34 Although they did not quantify the binding or assembly energies, they suggested that the assembly is mainly driven by electrostatic interactions between the protein and the nucleic acid. In contrast to this work, we attempt to understand the CP–cargo interactions not only qualitatively but also quantitatively, to gain understanding on the cargo-related aspects that stabilize or destabilize viruses and VLPs.…”

“…To this end, we have employed isothermal titration calorimetry (ITC) to monitor heat changes caused by the interaction between CP and cargo upon mixing. ITC has been used in viral research before, for example, to study membrane fusion events for the influenza virus, DNA ejection from bacteriophages, and the binding of inhibitors to viruses for the development of new antiviral treatments. , Moreover, ITC has been applied by De Souza et al to qualitatively study the assembly of hepatitis C virus core proteins around a nucleic acid . Although they did not quantify the binding or assembly energies, they suggested that the assembly is mainly driven by electrostatic interactions between the protein and the nucleic acid.…”