Molecular determinants for subcellular localization of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic alpha-helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-alpha. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.     

Regulated processing of hepatitis C virus core protein is linked to subcellular localization.

Posttranslational processing and subcellular localization of the HCV core protein are critical steps involved in the assembly of hepatitis C virus (HCV). In this study, both of these events were investigated by in vitro translation and transient COS-1 cell transfection of core protein expression constructs. Mutations at amino acid residues 173 to 174 and 191 to 192 disrupted processing events at the two putative cleavage sites in the C-terminal hydrophobic region of the core protein, indicating that these residues are implicated in the pathway of core protein maturation. As a result, two forms of core protein, C173 and C191, were detected by immunoblotting. Indirect immunofluorescence experiments showed that core proteins C173 and C191, when produced from HCV full-length protein or various polyprotein precursors, displayed a cytoplasmic localization. The C173 species, however, was translocated to the nucleus when expressed in the absence of C191. These findings indicate that preferential cleavage may occur during core protein maturation and that the association of the C191 with the C173 species may contribute to the distinct subcellular distribution of core protein. This may provide a possible mechanism for the control of the diverse biological functions of core protein during HCV replication and assembly.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed inE. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCI density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Fcgamma receptor-like activity of hepatitis C virus core protein

We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.     

Trafficking of hepatitis C virus core protein during virus particle assembly

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.     

Identification of a C-terminal regulatory motif in hepatitis C virus RNA-dependent RNA polymerase: structural and biochemical analysis

The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-A resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to approximately 50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.     

Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.     

Structural determinants that target the hepatitis C virus core protein to lipid droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.     

Immune responses to plasmid DNA encoding the hepatitis C virus core protein.

Hepatitis C virus (HCV) is a major causative agent of parenterally transmitted non-A, non-B hepatitis. The genomic region encoding the virion-associated core protein is relatively conserved among HCV strains. To generate a DNA vaccine capable of expressing the HCV core protein, the genomic region encoding amino acid residues 1 to 191 of the HCV-1 strain was amplified and cloned into an eukaryotic expression vector. Intramuscular inoculation of recombinant plasmid DNA into BALB/c mice (H-2d) generated core-specific antibody responses, lymphoproliferative responses, and cytotoxic T-lymphocyte activity. Our results suggest that the HCV core polynucleotide warrants further investigation as a potential vaccine against HCV infection.     

Suppression of ceramide-induced cell death by hepatitis C virus core protein

The hepatitis C virus (HCV) core protein is believed to be one of viral proteins that are capable of preventing virus-infected cell death upon various stimuli. But, the effect of the HCV core protein on apoptosis that is induced by various stimuli is contradictory. We examined the possibility that the HCV core protein affects the ceramide-induced cell death in cells expressing the HCV core protein through the sphingomyelin pathway. Cell death that is induced by C(2)-ceramide and bacterial sphingomyelinase was analyzed in 293 cells that constitutively expressed the HCV core protein and compared with 293 cells that were stably transfected only with the expression vector. The HCV core protein inhibited the cell death that was induced by these reagents. The protective effects of the HCV core protein on ceramide-induced cell death were reflected by the reduced expression of p21(WAF1/Cip1/Sid1) and the sustained expression of the Bcl-2 protein in the HCV core-expressing cells with respect to the vector-transfected cells. These results suggest that the HCV core protein in 293 cells plays a role in the modulation of the apoptotic response that is induced by ceramide. Also, the ability of the HCV core protein to suppress apoptosis might have important implications in understanding the pathogenesis of the HCV infection.     

Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein

Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.     

Synthesis of a novel hepatitis C virus protein by ribosomal frameshift.

Hepatitis C virus (HCV) is an important human pathogen that affects approximately 100 million people worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. We report here the discovery of a novel HCV protein synthesized by ribosomal frameshift. This protein, which we named the F protein, is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frameshift into the -2/+1 reading frame. This ribosomal frameshift requires only codons 8-14 of the core protein-coding sequence, and the shift junction is located at or near codon 11. An F protein analog synthesized in vitro reacted with the sera of HCV patients but not with the sera of hepatitis B patients, indicating the expression of the F protein during natural HCV infection. This unexpected finding may open new avenues for the development of anti-HCV drugs.     

Regulation of hepatitis C virus replication by the core protein through its interaction with viral RNA polymerase

The hepatitis C virus (HCV) core protein is a structural component of the nucleocapsid and has been shown to modulate cellular signaling pathways by interaction with various cellular proteins. In the present study, we investigated the role of HCV core protein in viral RNA replication. Immunoprecipitation experiments demonstrated that the core protein binds to the amino-terminal region of RNA-dependent RNA polymerase (RdRp), which encompasses the finger and palm domains. Direct interaction between HCV RdRp and core protein led to inhibition of RdRp RNA synthesis activity of in vitro. Furthermore, over-expression of core protein, but not its derivatives lacking the RdRp-interacting domain, suppressed HCV replication in a hepatoma cell line harboring an HCV subgenomic replicon RNA. Collectively, our results suggest that the core protein, through binding to RdRp and inhibiting its RNA synthesis activity, is a viral regulator of HCV RNA replication.     

Resveratrol prevents hepatic steatosis induced by hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the development of hepatic steatosis in patients with chronic HCV infection. Treatment of C57BL/6 mice infected with HCV core recombinant adenoviruses with resveratrol significantly decreased hepatic triacylglycerols (TAG) while the serum TAG level was unaffected. RT-PCR and Western blotting showed that HCV core protein attenuated the expression of Sirt1 and PPAR-α, which would be reversed by resveratrol. This was also confirmed in primary mouse hepatic cells infected with HCV core protein expressing adenovirus. Thus, resveratrol may prevent against hepatic steatosis by blocking the inhibited expression of Sirt1 and PPAR-α induced by HCV core protein.     

Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.     

Interaction between hepatitis C virus core protein and E1 envelope protein.

Hepatitis C virus has three structural genes named C, E1, and E2. The C gene encodes the core (capsid) protein and the E1 and E2 genes encode the envelope proteins. In an immunoprecipitation experiment, the E1 protein was found to be precipitated by an anti-core antibody in the presence but not in the absence of the core protein, indicating that the E1 protein can interact with the core protein. This interaction is independent of whether the E1 and the C genes are linked in cis or separated in different DNA constructs for expression. The interaction between the core and the E1 proteins is confirmed by the observation that a hybrid protein derived from the core protein and the tissue plasminogen activator is localized in the nucleus in the absence of the E1 protein and in the perinuclear region in the presence of the E1 protein. Deletion-mapping studies indicate that the carboxy-terminal sequences of both the core and the E1 proteins are important for their interaction. Since little E1 sequence is exposed on the cytosolic side of the membrane of the endoplasmic reticulum, the interaction between the core and the E1 proteins most likely takes place in the endoplasmic reticulum membrane. The E2 protein could not be coprecipitated with the core protein by the anti-core antibody in a similar assay and likely does not interact with the core protein. The implications of these findings on the morphogenesis of the hepatitis C virus virion are discussed.     

Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein

We have demonstrated previously that the core protein of hepatitis C virus (HCV) exhibits suppression activity on gene expression and replication of hepatitis B virus (HBV). Here we further elucidated the suppression mechanism of HCV core protein. We demonstrated that HCV core protein retained the inhibitory effect on HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the bipartite nuclear localization signal of the HCV core protein resulted in the cytoplasmic localization of core protein but did not affect its suppression ability on HBV gene expression. Mutational studies also indicated that almost all dibasic residue mutations within the N-terminal 101-amino acid segment of the HCV core protein (except Arg(39)-Arg(40)) impaired the suppression activity on HBV replication but not HBV gene expression. The integrity of Arg residues at positions 101, 113, 114, and 115 was found to be essential for both suppressive effects, whereas the Arg residue at position 104 was important only in the suppression of HBV gene expression. Moreover, our results indicated that the suppression on HBV gene expression was mediated through the direct interaction of HCV core protein with the trans-activator HBx protein, whereas the suppression of HBV replication involved the complex formation between HBV polymerase (pol) and the HCV core protein, resulting in the structural incompetence for the HBV pol to bind the package signal and consequently abolished the formation of the HBV virion. Altogether, this study suggests that these two suppression effects on HBV elicited by the HCV core protein likely depend on different structural context but not on nuclear localization of the core protein, and the two effects can be decoupled as revealed by its differential targets (HBx or HBV pol) on these two processes of the HBV life cycle.