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.     

Dynamics of lipid droplets induced by the hepatitis C virus core protein

The hepatitis C virus (HCV) is a global health problem, with limited treatment options and no vaccine available. HCV uses components of the host cell to proliferate, including lipid droplets (LD) onto which HCV core proteins bind and facilitate viral particle assembly. We have measured the dynamics of HCV core protein-mediated changes in LDs and rates of LD movement on microtubules using a combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), and differential interference contrast (DIC) microscopies. Results show that the HCV core protein induces rapid increases in LD size. Particle tracking experiments show that HCV core protein slowly affects LD localization by controlling the directionality of LD movement on microtubules. These dynamic processes ultimately aid HCV in propagating and the molecules and interactions involved represent novel targets for potential therapeutic intervention.     

GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.

GB virus B (GBV-B) is a recently discovered virus responsible for hepatitis in tamarins (Saguinus species). GBV-B belongs to the Flaviviridae family and is closely related to the human pathogen hepatitis C virus (HCV). Nonstructural protein 3 (NS3) of HCV has been shown to encompass a serine protease domain required for viral maturation. GBV-B and HCV share only about 30% of the amino acid sequence within the NS3 protease domain. The catalytic triad is conserved, and the residue Phe-154, presumed to be a crucial amino acid for determining the S1 specificity pocket of the HCV NS3 protease, is also conserved. We have expressed a synthetic gene encoding the GBV-B NS3 protease domain in Escherichia coli and have characterized the purified recombinant protein for its activity on HCV substrates. We have shown that the NS3 region of the GBV-B genome actually encodes a serine protease that, despite the low sequence homology, shares substrate specificity with the HCV NS3 protease.     

The birth and life of lipid droplets: learning from the hepatitis C virus

LDs (lipid droplets) are probably the least well-characterized cellular organelles. Having long been considered simple lipid storage depots, they are now considered to be dynamic organelles involved in many biological processes. However, most of the mechanisms driving LDs biogenesis, growth and intracellular movement remain largely unknown. As for other cellular mechanisms deciphered through the study of viral models, HCV (hepatitis C virus) is an original and relevant model for investigations of the birth and life of these organelles. Recent studies in this model have raised the hypothesis that the HCV core protein induces the redistribution of LDs through the regression and regeneration of these organelles in specific intracellular domains.     

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.     

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     

Phosphorylation of hepatitis B virus precore and core proteins.

Hepatitis B virus precore and core proteins are related. The precore protein contains the entire sequence of the core protein plus an amino-terminal extension of 29 amino acids. The amino-terminal extension of the precore protein contains a signal sequence for the secretion of the precore protein. This signal sequence is removed after the translocation of the precore protein across the endoplasmic reticulum membrane to produce the precore protein derivative named P22. We demonstrate that both P22 and the core protein can be phosphorylated in cells. Microsomal fractionation and trypsin digestion experiments demonstrate that a fraction of phosphorylated P22 is located in the endoplasmic reticulum lumen. Phosphorylation of P22 likely occurs in the carboxy terminus, since the P22 derivative P16, which lacks the carboxy terminus of P22, is not phosphorylated. Linking the carboxy terminus of the precore-core protein to heterologous secretory and cytosolic proteins led to the phosphorylation of the resulting chimeric proteins. These results indicate that phosphorylation of P22 and the core protein is likely mediated by cellular kinases.     

Conformational features of truncated hepatitis C virus core protein in virus-like particles

HCVc 120 is a truncated protein from the hepatitis C virus (HCV) core protein that interacts with itself to form nucleocapsid-like particles. We present here the infrared and Raman spectra of oligomeric HCVc 120 protein in order to obtain insights into its secondary structure as well as the environment surrounding some protein side chains. When compared with its monomer form, oligomeric HCVc 120 protein shows an increase in beta-sheet structure. Tryptophan residues have been found to be solvent exposed in the oligomeric form, and they likely do not significantly participate in the protein assembly. However, the beta-sheet content in oligomeric HCVc 120 protein suggests that this structural motif cannot be excluded in nucleocapsid formation, as shown recently in other viruses.     

Maturation of hepatitis C virus core protein by signal peptide peptidase is required for virus production

Complete maturation of hepatitis C virus (HCV) core protein requires coordinate cleavage by signal peptidase and an intramembrane protease, signal peptide peptidase. We show that reducing the intracellular levels of signal peptide peptidase lowers the titer of infectious virus released from cells, indicating that it plays an important role in virus production. Proteolysis by the enzyme at a signal peptide between core and the E1 glycoprotein is needed to permit targeting of core to lipid droplets. From mutagenesis studies, introducing mutations into the core-E1 signal peptide delayed the appearance of signal peptide peptidase-processed core until between 48 and 72 h after the beginning of the infectious cycle. Accumulation of mature core at these times coincided with its localization to lipid droplets and a rise in titer of infectious HCV. Therefore, processing of core by signal peptide peptidase is a critical event in the virus life cycle. To study the stage in virus production that may be blocked by interfering with intramembrane cleavage of core, we examined the distribution of viral RNA in cells harboring the core-E1 signal peptide mutant. Results revealed that colocalization of core with HCV RNA required processing of the protein by signal peptide peptidase. Our findings provide new insights into the sequence requirements for proteolysis by signal peptide peptidase. Moreover, they offer compelling evidence for a function for an intramembrane protease to facilitate the association of core with viral genomes, thereby creating putative sites for assembly of nascent virus particles.     

The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly

Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6- and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly.     

Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus.

Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes.     

Higher plant 5S rRNAs share common secondary and tertiary structure. A new three domains model

A new model of secondary and tertiary structure of higher plant 5S RNA is proposed. It consists of three helical domains: domain alpha includes stem I; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. Except for, presumably, a canonical RNA-A like domain alpha, the two remaining domains apparently adopt a perturbed RNA-A structure due to irregularities within internal loops B and E and three bulges occurring in the model. Bending of RNA could bring loops B and E and/or C and D closer making tertiary interactions likely. The model differs from that suggested for eukaryotic 5S rRNA, by organization of domain gamma. Our model is based on the results of partial digestion obtained with single- and double-strand RNA specific nucleases. The proposed secondary structure is strongly supported by the observation that crude plant 5S rRNA contains abundant RNA, identified as domain gamma of 5S rRNA. Presumably it is excised from the 5S rRNA molecule by a specific nuclease present in lupin seeds. Experimental results were confirmed by computer-aided secondary structure prediction analysis of all higher plant 5S rRNAs. Differences observed between earlier proposed models and our proposition are discussed.     

Identification of the domains required for the localization of Prp19p to lipid droplets or the nucleus

Prp19p is a protein found in the nucleus, cytosol or lipid droplets depending on the cell type. Prp19p participates in pre-mRNA splicing, in neuronal/astroglial cell fate decisions or in adipocyte lipid droplet biogenesis. In this study, the motifs of Prp19p that are necessary for its localization to lipid droplets or the nucleus in 3T3-L1 adipocytes are investigated using a series of truncated mutants of Prp19p that were fused to EGFP and transiently introduced into differentiated 3T3-L1 adipocytes. Immunofluorescence microscopy revealed that a domain of amino acids 167–250 is necessary for the recruitment of Prp19p to lipid droplets and that a domain of amino acids 1–166 is necessary for the recruitment of Prp19p to a nucleus.     

The central domain is required to target and anchor perilipin A to lipid droplets

The perilipins are the most abundant proteins coating the surfaces of lipid droplets in adipocytes and are found at lower levels surrounding lipid droplets in steroidogenic cells. Perilipins drive triacylglycerol storage in adipocytes by regulating the rate of basal lipolysis and are also required to maximize hormonally stimulated lipolysis. To map the domains that target and anchor perilipin A to lipid droplets, we stably expressed fragments of perilipin A in 3T3-L1 fibroblasts. Immunofluorescence microscopy and immunoblotting of proteins from isolated lipid droplets revealed that neither the amino nor the carboxyl terminus is required to target perilipin A to lipid droplets; however, there are multiple, partially redundant targeting signals within a central domain including 25% of the primary amino acid sequence. A peptide composed of the central domain of perilipin A directed a fused green fluorescent protein to the surfaces of lipid droplets. Full-length perilipin A associates with lipid droplets via hydrophobic interactions, as shown by the persistence of perilipins on lipid droplets after centrifugation through an alkaline carbonate solution. Results of the mutagenesis studies indicate that the sequences responsible for anchoring perilipin A to lipid droplets are most likely domains of moderately hydrophobic amino acids located within the central 25% of the protein. Thus, we conclude that the central 25% of the perilipin A sequence contains all of the amino acids necessary to target and anchor the protein to lipid droplets.