Detection of hepatitis C virus core protein circulating within different virus particle populations

Abstract

Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.

Direct Binding of Hepatitis C Virus Core to gC1qR on CD4+and CD8+T Cells Leads to Impaired Activation of Lck and Akt

“…Furthermore, dendritic cell maturation and, correspondingly, their CD4 ϩ -T-cellpriming ability are impaired by the HCV core, leading to a defect in the induction of anti-HCV T cells (36,37). Importantly, free core protein (non-virion associated) is secreted from infected cells and is detectable in the bloodstream of HCV-infected patients, possibly providing the virus with an indirect means of affecting host immunity (2,25,26,43). This free core protein has been shown to interfere with both proliferation and effector activities of human T cells through its interaction with a complement receptor, gC1qR, in a mixedlymphocyte reaction (18,44,45,46).…”

“…Intriguingly, the HCV core protein is capable of being secreted from infected cells and is therefore detectable in the bloodstream of HCV-infected patients (25,26). Free core protein, which is not associated with immune complexes or with virus particles, is especially detectable in the serologically negative window phase; the detection of total core protein (i.e., core-immune complex) in serum or plasma serves as an indirect marker for HCV replication and viremia (2,43).…”

Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation

“…It is notable that the minimal concentration of core protein that inhibits the Tcell proliferative responses is in the nanomolar range (1.3 nM) and may be comparable to the concentration of core protein detectable in the plasma of HCV-infected patients (34). In contrast, neither the addition of recombinant HCV NS3, one of HCV nonstructural proteins prepared in an identical manner as recombinant core, nor the addition of β-gal protein to MLR culture affected T-cell proliferation (Figure 5b).…”

“…While core protein is presumed to be normally present in the circulation of infected patients as the nucleocapsid component of intact HCV virions, the exposure of extra virion-free core protein in the blood and tissues is less clear. Core protein has been reported to be secreted from cells expressing the core gene (24) and free (nonvirion-associated) core protein has been detected in the plasma of HCV-infected patients (34). We have also readily detected core proteins in the plasma of several HCV-infected patients and HCV core transgenic mice without previous detergent treatment of the plasma (Y.S.…”

Hepatitis C Virus Core Protein Inhibits Human T Lymphocyte Responses by a Complement-Dependent Regulatory Pathway

“…Importantly, there is accumulating evidence that supports the presence of circulating core protein in the blood of HCV-infected patients. Core protein has been shown to be secreted from transfected cell lines expressing the core gene (25), and circulating core protein is detectable in the plasma of HCV-infected patients (26). These results suggest that core-induced immune suppression may play a critical role in establishing and maintaining HCV persistence during early viral infection.…”