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1.
Rodney K. Lyn Graham Hope Allison R. Sherratt John McLauchlan John Paul Pezacki 《PloS one》2013,8(11)
Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV. 相似文献
2.
Boulant S Douglas MW Moody L Budkowska A Targett-Adams P McLauchlan J 《Traffic (Copenhagen, Denmark)》2008,9(8):1268-1282
Attachment of hepatitis C virus (HCV) core protein to lipid droplets (LDs) is linked to release of infectious progeny from infected cells. Core progressively coats the entire LD surface from a unique site on the organelle, and this process coincides with LD aggregation around the nucleus. We demonstrate that LD redistribution requires only core protein and is accompanied by reduced abundance of adipocyte differentiation-related protein (ADRP) on LD surfaces. Using small hairpin RNA technology, we show that knock down of ADRP has a similar phenotypic effect on LD redistribution. Hence, ADRP is crucial to maintain a disperse intracellular distribution of LDs. From additional experimental evidence, LDs are associated with microtubules and aggregate principally around the microtubule-organizing centre in HCV-infected cells. Disrupting the microtubule network or microinjecting anti-dynein antibody prevented core-mediated LD redistribution. Moreover, microtubule disruption reduced virus titres, implicating transport networks in virus assembly and release. We propose that the presence of core on LDs favours their movement towards the nucleus, possibly to increase the probability of interaction between sites of HCV RNA replication and virion assembly. 相似文献
3.
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. 相似文献
4.
The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells. 相似文献
5.
Lo?c Etienne Emmanuelle Blanchard Audrey Boyer Virginie Desvignes Julien Gaillard Jean-Christophe Meunier Philippe Roingeard Christophe Hourioux 《PloS one》2015,10(9)
Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis. 相似文献
6.
Oshiumi H Ikeda M Matsumoto M Watanabe A Takeuchi O Akira S Kato N Shimotohno K Seya T 《PloS one》2010,5(12):e14258
The DEAD box helicase DDX3 assembles IPS-1 (also called Cardif, MAVS, or VISA) in non-infected human cells where minimal amounts of the RIG-I-like receptor (RLR) protein are expressed. DDX3 C-terminal regions directly bind the IPS-1 CARD-like domain as well as the N-terminal hepatitis C virus (HCV) core protein. DDX3 physically binds viral RNA to form IPS-1-containing spots, that are visible by confocal microscopy. HCV polyU/UC induced IPS-1-mediated interferon (IFN)-beta promoter activation, which was augmented by co-transfected DDX3. DDX3 spots localized near the lipid droplets (LDs) where HCV particles were generated. Here, we report that HCV core protein interferes with DDX3-enhanced IPS-1 signaling in HEK293 cells and in hepatocyte Oc cells. Unlike the DEAD box helicases RIG-I and MDA5, DDX3 was constitutively expressed and colocalized with IPS-1 around mitochondria. In hepatocytes (O cells) with the HCV replicon, however, DDX3/IPS-1-enhanced IFN-beta-induction was largely abrogated even when DDX3 was co-expressed. DDX3 spots barely merged with IPS-1, and partly assembled in the HCV core protein located near the LD in O cells, though in some O cells IPS-1 was diminished or disseminated apart from mitochondria. Expression of DDX3 in replicon-negative or core-less replicon-positive cells failed to cause complex formation or LD association. HCV core protein and DDX3 partially colocalized only in replicon-expressing cells. Since the HCV core protein has been reported to promote HCV replication through binding to DDX3, the core protein appears to switch DDX3 from an IFN-inducing mode to an HCV-replication mode. The results enable us to conclude that HCV infection is promoted by modulating the dual function of DDX3. 相似文献
7.
Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A 总被引:2,自引:0,他引:2
The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A. 相似文献
8.
Sofia C. Guimaraes Martin Schuster Ewa Bielska Gulay Dagdas Sreedhar Kilaru Ben R.A. Meadows Michael Schrader Gero Steinberg 《The Journal of cell biology》2015,211(5):945-954
Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell. 相似文献
9.
Hepatitis C virus core protein potentiates TNF-alpha-induced NF-kappaB activation through TRAF2-IKKbeta-dependent pathway 总被引:2,自引:0,他引:2
Chung YM Park KJ Choi SY Hwang SB Lee SY 《Biochemical and biophysical research communications》2001,284(1):15-19
Previous work has implicated that the core protein of hepatitis C virus (HCV) may play a modulatory effect on NF-kappaB activation induced by TNF-alpha. However, it is unclear how HCV core protein modulates TNF-alpha-induced NK-kappaB activation. Here we show that overexpression of HCV core protein potentiates NF-kappaB activation induced by TNF-alpha. Expression of dominant negative form of TRAF2 inhibits the synergistic effects of HCV core protein on NF-kappaB activation, suggesting that HCV core protein potentiates NF-kappaB activation through TRAF2. Moreover, we demonstrate that HCV core protein potentiates TRAF2-mediated NF-kappaB activation via IKKbeta. In addition, HCV core protein associates with TNF-R1-TRADD-TRAF2 signaling complex, resulting in synergistically activation of NF-kappaB induced by TNF-alpha. Thus, these observations indicate that HCV core protein may play an important role in the regulation of the cellular inflammatory and immune responses through NF-kappaB. 相似文献
10.
Farzin Roohvand Patrick Maillard Jean-Pierre Lavergne Steeve Boulant Marine Walic Ursula Andréo Lucie Goueslain Fran?ois Helle Adeline Mallet John McLauchlan Agata Budkowska 《The Journal of biological chemistry》2009,284(20):13778-13791
Early events leading to the establishment of hepatitis C virus (HCV)
infection are not completely understood. We show that intact and dynamic
microtubules play a key role in the initiation of productive HCV infection.
Microtubules were required for virus entry into cells, as evidenced using
virus pseudotypes presenting HCV envelope proteins on their surface. Studies
carried out using the recent infectious HCV model revealed that microtubules
also play an essential role in early, postfusion steps of the virus cycle.
Moreover, low concentrations of vinblastin and nocodazol,
microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules,
inhibited infection, suggesting that microtubule dynamic instability and/or
treadmilling mechanisms are involved in HCV internalization and early
transport. By protein chip and direct core-dependent pull-down assays,
followed by mass spectrometry, we identified β- and α-tubulin as
cellular partners of the HCV core protein. Surface plasmon resonance analyses
confirmed that core directly binds to tubulin with high affinity via amino
acids 2-117. The interaction of core with tubulin in vitro promoted
its polymerization and enhanced the formation of microtubules. Immune electron
microscopy showed that HCV core associates, at least temporarily, with
microtubules polymerized in its presence. Studies by confocal microscopy
showed a juxtaposition of core with microtubules in HCV-infected cells. In
summary, we report that intact and dynamic microtubules are required for virus
entry into cells and for early postfusion steps of infection. HCV may exploit
a direct interaction of core with tubulin, enhancing microtubule
polymerization, to establish efficient infection and promote virus transport
and/or assembly in infected cells.HCV5 infection is a
major cause of chronic liver disease, which frequently progresses to cirrhosis
and hepatocellular carcinoma. HCV represents a global public health problem,
with 130 million people infected worldwide. There is currently no vaccine
directed against HCV and the available antiviral treatments eliminate the
virus in 40-80% of patients, depending on the virus genotype (for review, see
Ref. 1).HCV has a single-stranded, positive-sense RNA genome of ∼9.6 kilobases
encoding a large polyprotein that is processed by both host and viral
proteases to produce three structural proteins (core protein and the envelope
glycoproteins E1 and E2), p7, and six nonstructural proteins, which are
involved in polyprotein processing and replication of the virus genome (for
review, see Ref. 2).HCV core is a basic protein, synthesized as the most N-terminal component
of the polyprotein, and is followed by the signal sequence of the E1 envelope
glycoprotein (3). The
polypeptide is cleaved by signal peptidase and signal peptide peptidase,
resulting in the release of core from the endoplasmic reticulum membrane and
its trafficking to lipid droplets
(3-5).
Mature core protein forms the viral nucleocapsid
(6) and consists of two
domains, D1 and D2. D1 lies at the protein N terminus, is composed of about
117 amino acids (aa), and is involved in RNA binding
(7). D2 is relatively
hydrophobic, has a length of about 55 aa, and targets HCV core to lipid
droplets (8).Microtubules (MTs) are ubiquitous cytoskeleton components that play a key
role in various cellular processes relating to cell shape and division,
motility, and intracellular trafficking
(9). MTs are dynamic, polarized
polymers composed of α/β-tubulin heterodimers that undergo
alternate phases of growth and shrinkage, dependent on so-called
“dynamic instability”
(10). Active transport by MTs
is bidirectional and involves both plus and minus end-directed motors: kinesin
and dynein (11,
12).Another mechanism of cytosolic transport on MTs, called
“treadmilling”
(13,
14) involves polymerization at
the plus end and depolymerization at the minus end after severing of MTs by
cellular katenin (15).MTs have important functions in the life cycle of most viruses
(13,
16,
17). Cytoplasmic transport on
MTs provides viruses with the means to reach sites of replication or enables
progeny virus to leave the infected cell. Some viruses, such as Ebola virus
(18) or reovirus
(19), are transported on MTs
within membranous compartments, whereas other viruses like herpes simplex
virus type 1 (20), murine
polyoma virus (21), human
cytomegalovirus (22), or
adenovirus (23) interact with
MT motors or MT-associated proteins to allow their transport along
microtubules.Previous studies have established that the cell cytoskeleton is involved in
HCV replication, since HCV replication complexes are subjected to
intracellular transport and their formation is closely linked to the dynamic
organization of endoplasmic reticulum, actin filaments, and the microtubule
network
(24-26).
In addition, intact microtubules are essential for viral morphogenesis and the
secretion of progeny virus from infected cells
(27). The role of microtubules
in HCV cell entry and the initiation of productive HCV infection has not yet
been addressed.In this study, we provide evidence that the MT network plays a key role in
HCV cell entry and postfusion steps of the virus cycle that lead to the
establishment of productive HCV infection. The initial steps of the viral
cycle are sensitive to MT-affecting drugs that inhibit MT formation or
depolymerize or stabilize microtubules. We also show a unique property of the
HCV core protein, its capacity to directly bind to tubulin and to enhance MT
polymerization in vitro. Our findings suggest that HCV could exploit
the MT network by polymerization-related mechanisms to productively infect its
target cell. Thus, microtubules may provide a novel target for therapeutic
interventions against HCV infection. 相似文献
11.
Yoshihiro Matsumoto Tomokazu Matsuura Haruyo Aoyagi Mami Matsuda Su Su Hmwe Tomoko Date Noriyuki Watanabe Koichi Watashi Ryosuke Suzuki Shizuko Ichinose Kenjiro Wake Tetsuro Suzuki Tatsuo Miyamura Takaji Wakita Hideki Aizaki 《PloS one》2013,8(7)
Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release. 相似文献
12.
Maillard P Lavergne JP Sibéril S Faure G Roohvand F Petres S Teillaud JL Budkowska A 《The Journal of biological chemistry》2004,279(4):2430-2437
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. 相似文献
13.
Miyanari Y Atsuzawa K Usuda N Watashi K Hishiki T Zayas M Bartenschlager R Wakita T Hijikata M Shimotohno K 《Nature cell biology》2007,9(9):1089-1097
The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions. 相似文献
14.
Hepatitis C Virus Genotype 1b Core Protein Does Not Exert Immunomodulatory Effects on Virus-Induced Cellular Immunity 总被引:3,自引:0,他引:3
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Zhang-Xu Liu Hiroshi Nishida Jian-Wen He Michael M. C. Lai Ni Feng Gunther Dennert 《Journal of virology》2002,76(3):990-997
The hepatitis C virus (HCV) core protein is among the most conserved proteins in HCV and is known to induce sensitization of cytotoxic T lymphocytes (CTL). Therefore, it is a prime candidate for a component of a potential HCV vaccine. The HCV core protein has, however, been reported to exert multiple effects on cell functions, raising questions as to its suitability for this purpose. This question was investigated here with mice into which replication-deficient adenoviruses expressing core protein of an HCV genotype 1b isolate were injected. We show that induction of cytokines in response to the infection, infiltration of lymphocytes into the infected liver, priming of virus-specific CTL, and liver injury are not modulated by expression of the core protein in the liver. Moreover, no changes in the sensitivity to tumor necrosis factor alpha- or Fas-mediated liver injury are demonstrable. A similar lack of demonstrable effects of the core protein on immune functions has also been obtained using transgenic mice expressing another HCV genotype 1b core protein. It is concluded that the HCV core protein of genotype 1b has no modulatory effects on induction of virus-specific immune responses and may therefore be a suitable component of an HCV vaccine. 相似文献
15.
16.
Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features 总被引:3,自引:0,他引:3
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The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (C(HCV)169) and its N-terminal region from positions 1 to 117 (C(HCV)117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins C(HCV)169 and C(HCV)117 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (C(HCV)169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of alpha-helices (50%). In contrast, C(HCV)117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro. 相似文献
17.
The Native Form and Maturation Process of Hepatitis C Virus Core Protein 总被引:23,自引:5,他引:23
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Kohichiroh Yasui Takaji Wakita Kyoko Tsukiyama-Kohara Shin-Ichi Funahashi Masumi Ichikawa Tadahiro Kajita Darius Moradpour Jack R. Wands Michinori Kohara 《Journal of virology》1998,72(7):6048-6055
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. 相似文献
18.
Cerutti A Maillard P Minisini R Vidalain PO Roohvand F Pecheur EI Pirisi M Budkowska A 《PloS one》2011,6(10):e25854
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. 相似文献
19.
20.
Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle. 相似文献