Complex formation between hepatitis C virus core protein and p21Waf1/Cip1/Sdi1

The core protein (Core) of hepatitis C virus (HCV) has been known to play an important role in hepatocarcinogenesis. By using glutathione S-transferase (GST) pull-down assay, we show here that Core formed a complex with p21Waf1/Cip1/Sdi1 (p21) cell cycle regulator. The deletion-mapping analysis revealed that a portion near the N-terminus of Core (amino acids 24-52) and a C-terminal portion of p21 (amino acids 139-164) were involved in the complex formation. The complex formation was not impaired by point mutations of p21 at residues 147, 149, and 150, which have been reported to abrogate interaction of p21 with proliferating cell nuclear antigen (PCNA), discriminating the Core-binding sequence from the PCNA-binding sequence. Due to the close vicinity of the binding sites, however, Core and PCNA competed with each other when interacting with p21. The distinct interaction between Core and p21 may provide a new aspect to the studies of HCV pathogenesis.     

Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient

Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.     

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.     

The identification of three sizes of core proteins during the establishment of persistent hepatitis C virus infection in vitro

Similar to Hepatitis C virus (HCV) infection in humans, HCVcc infection can also result in persistent and chronic infection. The core protein is a variable protein and exists in several sizes. Some sizes of core proteins have been reported to be related to chronic HCV infection. To study the possible role of the core protein in persistent HCV infection, a persistent HCVcc infection was established, and the expression of the core protein was analysed over the course of the infection. The results show that there are three sizes of core proteins (p24, p21 and p19) expressed during the establishment of persistent HCVcc infection. Of these, the p21 core protein is the mature form of the HCV core protein. The p24 core protein is the phosphorylated form of p21. The p19 core protein appears to be a functional by-product generated during the course of infection. These three core proteins are all localized in the cytoplasm and can be encapsidated into the HCV virion. The appearance of the p19 and p24 core proteins might be related to acute HCVcc infection and chronic infection respectively and may play an important role in the pathology of a HCV infection.     

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

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.     

A concerted action of hepatitis C virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.     

Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Leu(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.     

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.     

Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast

To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1 , a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the γ-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21 , we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20 .     

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.     

Thr 446 phosphorylation of PKR by HCV core protein deregulates G2/M phase in HCC cells

Hepatitis C virus (HCV) is the major causative viral agent of cirrhosis and hepatocarcinoma (HCC). HCV core protein affects cell homeostasis, playing an important role in viral pathogenesis of HCC. We investigate the effects of HCV core protein expression on cell growth in HCC cell lines. Cell cycle distribution analysis of HepG2 polyclonal core positive cells reveals a peculiar accumulation of cells in G2/M phase. Different pathways mediate G2/M arrest: such as p53 and double strand RNA protein kinase (PKR). Flow cytometry in p53-null cells demonstrates that p53 plays only a marginal role in inducing HCV core-dependent G2/M phase accumulation that seems to be significantly affected by the functional inactivation of PKR. HCC core positive cells are characterized by a significant PKR phosphorylation in Thr 446 residue, which leads deregulation of mitosis. Moreover, we observe that the overexpression of the viral protein induces an upregulation of PKR activity, which does not correlate with an increased eIF-2 phosphorylation. This uncommon behavior of PKR suggests that its activation by HCV core protein could involve alternative PKR-dependent pathways, implicated in core-dependent G2/M accumulation. The described biological effects of HCV core protein on cell cycle could be an additional viral mechanism for both HCV resistance to interferon (IFN) and HCC HCV-related pathogenesis.     

Protein kinase B/Akt may regulate G2/M transition in the fertilized mouse egg by changing the localization of p21(Cip1/WAF1)

Protein kinase B (PKB, also called Akt) is known as a serine/threonine protein kinase. Some studies indicate that the Akt signalling pathway strongly promotes G2/M transition in mammalian cell cycle progression, but the mechanism remains to be clarified, especially in the fertilized mouse egg. Here, we report that the expression of Akt at both the protein and mRNA level was highest in G2 phase, accompanied by a peak of Akt activity. In addition, the subcellular localization of p21(Cip1/WAF1) has been proposed to be critical in the cell cycle. Hence, we detected the expression and localization of p21(Cip1/WAF1) after injecting fertilized mouse eggs with Akt mRNA. In one-cell stage fertilized embryos microinjected with mRNA coding for a constitutively active myristoylated Akt (myr-Akt), p21(Cip1/WAF1) was retained in the cytoplasm. Microinjection of mRNA of kinase-deficient Akt(Akt-KD) resulted in nuclear localization of p21(Cip1/WAF1) . Meanwhile, microinjection of different types of Akt mRNA affected the phosphorylation status of p21(Cip1/WAF1) . However, there was no obvious difference in the protein expression of p21(Cip1/WAF1) . Therefore, Akt controls the cell cycle by changing the subcellular localization of p21(Cip1/WAF1) , most likely by affecting the phosphorylation status of p21(Cip1/WAF1) .