Hepatitis c virus infection pdf

Annals of epidemiology. Changes in liver-related mortality by etiology and sequelae: underlying versus multiple causes of death. Population Health Metrics. View 1 excerpt, cites background. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. Harm Reduction Journal. JAMA network open. This cohort study evaluates the first 22 months of implementation of the Cherokee Nation Heath Services community-based program designed to eliminate hepatitis C virus HCV infection.

Inhibitory activity of limonoids from Khaya grandifoliola C. DC Meliaceae against hepatitis C virus infection in vitro. Avicenna journal of phytomedicine. View 2 excerpts, cites background. Morbidity and mortality weekly report. B shows a maximal intensity projection of a Z-series generated from a stack of 20 images.

To remove the blurring effect due to Z-stack acquisition, the Z-series was deconvolved using a blind deconvolution protocol. The image in C is a three-dimensional reconstruction of the zoomed area indicated by the white box. Microtubules in green and core protein in red are represented as solid isosurfaces. This assay is based on fluorescence enhancement due to the incorporation of a fluorescent reporter to MTs as polymerization occurs, generating a polymerization curve that represents the three phases of microtubule formation: nucleation, growth, and steady state equilibrium.

Compounds that interact with tubulin could alter one or more of these phases of polymerization. The addition of either of the two core proteins aa or aa to a tubulin polymerization reaction significantly increased the rate of microtubule polymerization to a similar extent as the antimitotic drug paclitaxel, which eliminates the nucleation phase and enhances the growth phase Fig. Adding vinblastin decreased the polymerization rate and caused significant reduction in polymer mass. Various concentrations of vinblastin induced a dose-dependent decrease of microtubule mass in vitro Fig.

The HCV core protein promotes polymerization of tubulin in vitro. The effect of the drug was monitored by a fluorescence assay as a function of time crosses. Tubulin polymerization in the absence of drug filled squares and in the presence of HCV core protein aa open triangles is shown. C , the HCV core protein and vinblastin were simultaneously added to the polymerization mixture. Tubulin polymerization in the absence of core filled squares and in the presence of core aa open triangles was assessed.

We examined further whether the HCV core could promote tubulin polymerization in the presence of vinblastin. In this series of experiments, different concentrations of vinblastin and a constant concentration of HCV core were used. The polymerization process was monitored by an in vitro polymerization assay in comparison with standard polymerization conditions. Nevertheless, in the presence of core, the effect of the drug was attenuated, as compared with that induced in the absence of core.

These observations are consistent with the potent effect of the HCV core protein on tubulin polymerization and the role of the N-terminal D1 domain of core in this process. HCV Core Protein Remains Associated with Polymerized Microtubules —To ascertain that, indeed, the HCV core protein enhances tubulin polymerization and induces formation of microtubules, MTs formed in vitro in the presence of core were analyzed by electron microscopy. This series of experiments demonstrated that MTs formed in the presence of core shown in Fig.

Strikingly, MTs polymerized in the presence of core bound anti-core antibodies, as ascertained by immune electron microscopy. These observations provided evidence that the HCV core enhances MT polymerization in the absence of other co-factors such as MT-associated proteins and associates, at least temporarily, with MTs Fig.

HCV core protein promotes tubulin polymerization and associates with MTs. A-D , control experiments to ascertain that MT polymerizes in the presence of core protein. E-G , immune electron microscopy analyses of microtubules polymerized in vitro in the presence of core protein. Microtubules formed in the presence of core protein aa E and F and in the presence of paclitaxel as negative control G. Arrows , colloidal gold beads. HCV is considered to enter the cell by clathrin-mediated endocytosis and fusion between the viral envelope and endosome membranes that occurs upon acidification of the endosomal compartment 35 , In this report using the JFH1 in vitro model, which reproduces a complete virus cell cycle, and drugs affecting main cytoskeleton components, we provide evidence that intact and dynamic MTs play a key role in the early steps of the virus cycle leading to the establishment of productive HCV infection.

Indeed, drugs that inhibit tubulin polymerization vinblastin or disrupt nocodazol or stabilize paclitaxel MTs inhibit also early steps of infection. In contrast, cytochalasin D, an inhibitor of actin polymerization and thus actin-dependent cellular transport, had no direct effect on the initiation of HCV infection. Using virus pseudotypes HCVpp as a model for studies of HCV cell entry 32 - 35 , we demonstrate that the first steps of virus internalization, from its attachment to the cell surface until fusion of the viral envelope glycoproteins within an endosomal compartment, depend on the intact MT network.

Our further studies carried out on the HCVcc model demonstrated also that early postentry steps of the virus cycle, after fusion of the virus envelope, which is estimated to be completed 2 h after the initiation of infection, require functional MTs. Indeed, a significant decrease in the production of the virus was observed when vinblastin was applied at various time points up to 8 h postinfection.

These data showed that an intact microtubule network is needed for early events of the HCV cell cycle. These events most probably also concern virus nucleocapsid release and early transport, subsequent to virus fusion. The effect of MT-affecting drugs is concentration-dependent and thus may reflect the mechanisms involved in virus interaction with MTs 40 , We observed the substantial effect of vinblastin and nocodazol on the initiation of HCV infection, when these drugs were used even in low micromolar and nanomolar concentrations.

Indeed, in micromolar concentrations, nocodazole induces depolymerization of MTs and tubulin aggregation. Nocodazol at low concentrations suppresses the association and dissociation rates of tubulin, stabilizing the MT dynamics, but does not alter the equilibrium between a MT polymer and soluble tubulin. Equally, nanomolar concentrations of vinblastin do not induce perceptible changes in MT structure; however, at these concentrations, vinblastin is known to suppress MT dynamic instability and treadmilling mechanisms.

Paclitaxel, an MT-stabilizing drug, also had a significant inhibitory effect on the establishment of productive HCV infection. Indeed, our observations using confocal microscopy evidenced juxtaposition of core with microtubules in HCV-infected cells. We equally provided evidence that the direct interaction of D1 of core with tubulin, without any mediators such as MT-associated proteins , promotes tubulin polymerization in vitro.

HCV core displayed an effect opposite to that of vinblastin, a drug that inhibits MT polymerization. When applied in the presence of vinblastin, core attenuated the inhibitory effect of the drug on MT polymerization.

Our observations by electron microscopy confirmed that microtubules formed in the presence of core are very similar to those formed in the presence of paclitaxel, a drug known to enhance microtubule polymerization Most importantly, immune electron microscopy analyses revealed that core protein remained associated with microtubules formed in vitro in its presence.

Viral genome-protein complexes, viruses, and subviral particles can be transported within the host cell cytoplasm during cell entry, from the plasma membrane to the site of viral replication, and during viral assembly and egress to the plasma membrane for their release into the extracellular milieu 13 , The association of viral proteins with microtubule components has been reported for several viruses: vaccinia virus 49 , herpes simplex virus 50 , murine coronavirus 51 , pseudorabies virus 52 , vesicular stomatitis virus 53 , rotavirus 54 , and M protein of Sendai virus During infection, some viruses are transported on MTs within membranous vesicles 18 , 19 , whereas others use mechanisms driven by microtubule motors for transport of their capsids or subviral particles on MTs interacting either with microtubule motors 20 - 23 or with microtubule-associated proteins Only three proteins have been shown up to now to directly interact with tubulin and to enhance tubulin polymerization in vitro : human immunodeficiency virus Tat protein 57 , tobacco mosaic virus movement protein 58 , and Ebola virus matrix protein VP40 Nevertheless, the exact mechanisms and role of these interactions in virus transport remain to be elucidated.

Transport of molecules along MTs can be achieved by using MT-dependent motors, such as kinesins and dyneins 11 , 12 , These motors use energy derived from ATP hydrolysis to move cargo, but motor-driven processes are not related to polymerization and depolymerization kinetics Thus, intracellular transport of the HCV core, similarly to the tobacco virus encoded movement protein or the human immunodeficiency virus Tat protein, which also enhance MT polymerization 57 , 58 , can be mediated by mechanisms related to tubulin polymerization.

HCV-RNA replication takes place in the cytoplasm in membrane-associated replication complexes designated as membranous webs The cytoskeleton components microtubules and actin filaments provide tracks for the movement of replication complexes 24 - Two types of HCV replication complexes have recently been reported; large structures, with restricted mobility appeared 24 h postinfection, and small replication complexes were formed h postinfection and moved in a MT-dependent manner These studies estimated that the formation of replication complexes starts about 12 h after the initiation of HCVcc infection, thus later than the early events analyzed in our study.

HCV morphogenesis and the secretion of progeny virus also require a functional microtubule network HCV core protein probably recruits nonstructural proteins and replication complexes to lipid droplet-associated membranes, and this process is directly connected to the virus assembly pathway in HCV-infected cells In HCV-infected cells, newly synthesized core protein loads lipid droplets and progressively coats the entire organelle, replacing adipocyte differentiation-related protein Core protein-coated lipid droplets are transported on microtubules in a dynein-dependent manner, h postinfection, to the perinuclear area, where virus morphogenesis takes place The above quoted studies focused either on the formation and transport of HCV replication complexes or on virus morphogenesis and release but did not address the role of microtubules in HCV cell entry and early transport.

Our study provides the first evidence that a microtubule network that is both intact and dynamic plays a key role in virus internalization, leading to the establishment of infection. Although many viruses use the microtubule network for virus transport at various steps of their life cycle, our study suggests that the establishment of productive HCV infection requires a dynamic process driven by microtubule polymerization.

Such mechanisms have been reported for a few viruses, such as duck hepatitis B virus or HHP-8 63 , Indeed, for these viruses, early steps of infection subsequent to the virus cell entry depend on both intact microtubules and their dynamic turnover.

Another possible function of the HCV core protein could be the regulation of dynein- and kinesin-based motions. Since bidirectional motion is widespread and direction of the microtubule-dependent transport depends on the balance between plus end and minus end directed motion, the cargo-based regulation of motor-MT interactions may be a key mechanism that is used to control cargo transport 11 , 12 , Proteins showing the ability to bind to MTs independently of motors could play a crucial role in such regulation In accordance with this notion, direct binding of core to tubulin and activation of microtubule polymerization could alter the balance of dynein- and kinesin-based motions.

Indeed, the regulation of motor complexes by virus capsid proteins has been suggested 65 , but the exact mechanism of such regulations has not yet been elucidated. Our findings show a novel and potentially important property of the core protein and suggest that HCV might exploit the MT lattice for transport of the virus nucleocapsid by polymerization-related mechanisms.

Such interactions could also play a role at later steps of the viral cycle, during viral morphogenesis and secretion. Improving our understanding of the consequences of core-MT interactions might facilitate the development of new therapeutic approaches targeted to inhibit HCV infection.

Indeed, molecules modifying MT dynamics are already an integral part of anti-cancer treatment We thank Y. Rouille for valuable discussion, T. Delagneau for ACAP monoclonal antibody. We express our gratitude to J. D'Alayer for help in protein chip and pull-down assays and E.

Perret in immunofluorescence microscopy. We are grateful to K. Kean for a critical review of the manuscript. National Center for Biotechnology Information , U.

Journal List J Biol Chem v. J Biol Chem. Author information Article notes Copyright and License information Disclaimer. Received Oct 14; Revised Mar 4. This article has been cited by other articles in PMC.

Abstract Early events leading to the establishment of hepatitis C virus HCV infection are not completely understood. Open in a separate window. Acknowledgments We thank Y. References 1. Pawlotsky, J. Penin, F. McLauchlan, J. How current direct-acting antiviral and novel cell culture systems for HCV are shaping therapy and molecular diagnosis of chronic HCV infection. Curr Drug Targets. Cell Host Microbe.

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