Kooperativer Bibliotheksverbund

Berlin Brandenburg

and
and

Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
Filter
  • Pietschmann, Thomas  (14)
Type of Medium
Language
Year
  • 1
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(7), p.e0134529
    Description: Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 2
    Language: English
    In: Hepatology (Baltimore, Md.), January 2014, Vol.59(1), pp.24-34
    Description: Hepatitis C Virus (HCV) entry involves at least four cellular factors, including CD81, the scavenger receptor class B type I (SCARB-1), occludin (OCLN), and claudin-1 (CLDN1). In addition, CLDN6 and CLDN9 have been shown to substitute for CLDN1 as HCV entry factors in human nonliver cells. We examined the role of different CLDN proteins during HCV entry by using cell lines expressing either predominantly CLDN1 (Huh-7.5) or CLDN6 (HuH6). Huh-7.5 cells were susceptible to all tested HCV isolates, whereas HuH6 cells were only permissive to some viral strains. Silencing of CLDN6 in HuH6 cells revealed that these cells are infected in a CLDN6-dependent fashion, and ectopic expression of CLDN1 or CLDN6 in 293T cells lacking endogenous CLDN expression confirmed that only some HCV strains efficiently use CLDN6 for infection. CLDN1-specific neutralizing antibodies (Abs) fully abrogated infection of Huh-7.5 cells by isolates that use CLDN1 only, whereas viruses with broad CLDN tropism were only partially inhibited by these Abs. Importantly, infection by these latter strains in the presence of anti-CLDN1 Ab was further reduced by silencing CLDN6, suggesting that viruses with broad CLDN usage escape CLDN1-specific Abs by utilization of CLDN6. Messenger RNA (mRNA) levels of HCV entry factors in liver biopsies of HCV patients infected with different genotype and with variable degree of liver fibrosis were determined. Uniformly high levels of CD81, SCARB-1, OCLN, and CLDN1 mRNA were detected. In contrast, abundance of CLDN6 mRNA was highly variable between patients. These findings highlight differential CLDN usage by HCV isolates, which may evolve based on variable expression of CLDN proteins in human liver cells. Broad CLDN tropism may facilitate viral escape from CLDN1-specific therapeutic strategies.
    Keywords: Viral Tropism ; Virus Internalization ; Claudin-1 -- Metabolism ; Claudins -- Metabolism ; Hepacivirus -- Physiology
    E-ISSN: 1527-3350
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 3
    Language: English
    In: Antiviral Research, 2011, Vol.89(2), pp.136-148
    Description: Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success. To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.
    Keywords: Hepatitis C Virus ; Chronic Liver Disease ; Hcv ; Viral Life Cycle ; Antiviral Therapy ; Tartrolon ; Medicine ; Biology
    ISSN: 0166-3542
    E-ISSN: 1872-9096
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 4
    In: Hepatology, January 2014, Vol.59(1), pp.24-34
    Description: Hepatitis C Virus (HCV) entry involves at least four cellular factors, including CD81, the scavenger receptor class B type I (SCARB‐1), occludin (OCLN), and claudin‐1 (CLDN1). In addition, CLDN6 and CLDN9 have been shown to substitute for CLDN1 as HCV entry factors in human nonliver cells. We examined the role of different CLDN proteins during HCV entry by using cell lines expressing either predominantly CLDN1 (Huh‐7.5) or CLDN6 (HuH6). Huh‐7.5 cells were susceptible to all tested HCV isolates, whereas HuH6 cells were only permissive to some viral strains. Silencing of CLDN6 in HuH6 cells revealed that these cells are infected in a CLDN6‐dependent fashion, and ectopic expression of CLDN1 or CLDN6 in 293T cells lacking endogenous CLDN expression confirmed that only some HCV strains efficiently use CLDN6 for infection. CLDN1‐specific neutralizing antibodies (Abs) fully abrogated infection of Huh‐7.5 cells by isolates that use CLDN1 only, whereas viruses with broad CLDN tropism were only partially inhibited by these Abs. Importantly, infection by these latter strains in the presence of anti‐CLDN1 Ab was further reduced by silencing CLDN6, suggesting that viruses with broad CLDN usage escape CLDN1‐specific Abs by utilization of CLDN6. Messenger RNA (mRNA) levels of HCV entry factors in liver biopsies of HCV patients infected with different genotype and with variable degree of liver fibrosis were determined. Uniformly high levels of CD81, SCARB‐1, OCLN, and CLDN1 mRNA were detected. In contrast, abundance of CLDN6 mRNA was highly variable between patients. : These findings highlight differential CLDN usage by HCV isolates, which may evolve based on variable expression of CLDN proteins in human liver cells. Broad CLDN tropism may facilitate viral escape from CLDN1‐specific therapeutic strategies. (H 2014;58:24–34)
    Keywords: Antibodies–Immunology ; Biopsy–Metabolism ; Cell Line, Tumor–Metabolism ; Claudin-1–Physiology ; Claudin-1–Metabolism ; Claudins–Pathology ; Hek293 Cells–Metabolism ; Hepacivirus–Metabolism ; Humans–Metabolism ; Liver–Metabolism ; Liver–Metabolism ; RNA, Messenger–Metabolism ; Viral Tropism–Metabolism ; Virus Internalization–Metabolism ; Hepatitis ; Hepatology ; Infections ; Medical Research ; Antibodies ; Cldn1 Protein, Human ; Claudin-1 ; Claudins ; RNA, Messenger ; Claudin 6;
    ISSN: 0270-9139
    E-ISSN: 1527-3350
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 5
    Language: English
    In: 2013, Vol.9(5), p.e1003355
    Description: Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7. ; Viroporins are small hydrophobic viral membrane proteins which oligomerize and modulate membrane properties to facilitate virus propagation. Within their membrane environment these proteins can form membrane pores or channels which change the permeability of membranes for ions. These properties are known to contribute to release of infectious enveloped virus particles from infected cells and/or to facilitate viral cell entry by catalyzing virus uncoating. In case of HCV, p7 function is essential for production of infectious progeny and its ion channel activity is well documented and in cell-based systems. Recent evidence indicated that p7 channel activity dissipates the low pH of the cellular secretory compartment thus protecting the viral glycoproteins from low pH induced misfolding and inactivation. In this investigation we highlight the involvement of the p7 ion channel in the assembly and envelopment of viral RNA-containing capsids. Our results indicate that p7, likely in concert with the viral envelope proteins, comprises a membrane-bound recipient complex that provides a scaffold to initiate unloading of core protein from lipid droplets for capsid assembly and membrane envelopment. Collectively, these findings highlight novel facets of p7 function in the course of HCV morphogenesis.
    Keywords: Research Article ; Biology
    ISSN: 1553-7366
    E-ISSN: 1553-7374
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 6
    Language: English
    In: PLoS Pathogens, 2012, Vol.8(7), p.e1002829
    Description: Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny. ; The human genome encodes more than 30 phospholipase A2s. These enzymes cleave fatty acids at the C2 atom of phosphoglycerides and thus modulate membrane properties. Among all PLA2s only PLA2G4A, which is recruited to perinuclear membranes by Ca and activated by extracellular stimuli via the mitogen activated protein kinase pathway, specifically cleaves lipids with arachidonic acid. Metabolism of arachidonic acid yields prostaglandins and leukotriens, important lipid mediators of inflammation. We show that inhibition of PLA2G4A produces aberrant HCV particles and that infectivity is rescued by addition of arachidonic acid. Our results suggest that a specific lipid (arachidonic acid) is essential for production of highly infectious HCV progeny, likely by creating a membrane environment conducive for efficient incorporation of crucial host and viral factors into the lipid envelope of nascent particles. Strikingly, PLA2G4A is also essential for production of highly infectious Dengue Virus (DENV) particles but not for vesicular stomatitis virus (VSV). These observations argue that HCV and DENV which unlike VSV produce particles at intracellular membranes usurp a common host factor (PLA2G4A) for assembly of highly infectious progeny. These findings open new perspectives for antiviral intervention and highlight thus far unrecognized parallels in the assembly pathway of HCV and DENV.
    Keywords: Research Article ; Biology ; Medicine ; Infectious Diseases ; Molecular Biology ; Cell Biology ; Gastroenterology And Hepatology
    ISSN: 1553-7366
    E-ISSN: 1553-7374
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 7
    Language: English
    In: PLoS Pathogens, 2018, Vol. 14(7)
    Description: Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells.
    Keywords: Medical And Health Sciences ; Basic Medicine ; Microbiology In The Medical Area ; Medicin Och Hälsovetenskap ; Medicinska Och Farmaceutiska Grundvetenskaper ; Mikrobiologi Inom Det Medicinska Området
    ISSN: 1553-7366
    E-ISSN: 15537374
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 8
    Language: English
    In: Journal of virology, February 2014, Vol.88(3), pp.1433-46
    Description: Hepatitis C virus (HCV) predominantly infects human hepatocytes, although extrahepatic virus reservoirs are being discussed. Infection of cells is initiated via cell-free and direct cell-to-cell transmission routes. Cell type-specific determinants of HCV entry and RNA replication have been reported. Moreover, several host factors required for synthesis and secretion of lipoproteins from liver cells, in part expressed in tissue-specific fashion, have been implicated in HCV assembly. However, the minimal cell type-specific requirements for HCV assembly have remained elusive. Here we report that production of HCV trans-complemented particles (HCVTCP) from nonliver cells depends on ectopic expression of apolipoprotein E (ApoE). For efficient virus production by full-length HCV genomes, microRNA 122 (miR-122)-mediated enhancement of RNA replication is additionally required. Typical properties of cell culture-grown HCV (HCVcc) particles from ApoE-expressing nonliver cells are comparable to those of virions derived from human hepatoma cells, although specific infectivity of virions is modestly reduced. Thus, apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTTP), and apolipoprotein C1 (ApoC1), previously implicated in HCV assembly, are dispensable for production of infectious HCV. In the absence of ApoE, release of core protein from infected cells is reduced, and production of extracellular as well as intracellular infectivity is ablated. Since envelopment of capsids was not impaired, we conclude that ApoE acts after capsid envelopment but prior to secretion of infectious HCV. Remarkably, the lack of ApoE also abrogated direct HCV cell-to-cell transmission. These findings highlight ApoE as a host factor codetermining HCV tissue tropism due to its involvement in a late assembly step and viral cell-to-cell transmission.
    Keywords: Viral Tropism ; Virus Assembly ; Apolipoproteins E -- Metabolism ; Hepacivirus -- Physiology ; Hepatitis C -- Metabolism
    ISSN: 0022538X
    E-ISSN: 1098-5514
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 9
    Language: English
    In: PLoS Pathogens, 2016, Vol. 12(4)
    Description: Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.
    Keywords: Medical And Health Sciences ; Basic Medicine ; Microbiology In The Medical Area ; Medicin Och Hälsovetenskap ; Medicinska Och Farmaceutiska Grundvetenskaper ; Mikrobiologi Inom Det Medicinska Området
    ISSN: 1553-7366
    E-ISSN: 15537374
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 10
    Language: English
    In: Journal of virology, November 2014, Vol.88(21), pp.12644-55
    Description: Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells by using at least four cell entry factors. These factors include scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1), and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interactions with these factors. Hypervariable region 1 (HVR1) within viral envelope protein 2 (E2) is involved in the usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage, and cell entry between wild-type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1, and OCLN usage. However, unlike wild-type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered the infection efficiencies of both viruses to similar levels. Analysis of affinity-purified virions revealed comparable levels of apolipoprotein E (ApoE) incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules, and it is critical only for wild-type HCV. The other one is important for both viruses but apparently is not inactivated by the SR-BI binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle-associated ApoE. HCV cell entry is SR-BI dependent irrespective of the presence or absence of HVR1. Moreover, this domain modulates the properties of ApoE on the surface of virus particles. These findings have implications for the development of SR-BI-targeting antivirals. Furthermore, these findings highlight separable functions of SR-BI during HCV cell entry and reveal a novel role of HVR1 for the properties of virus-associated lipoproteins.
    Keywords: Virus Internalization ; Claudin-1 -- Metabolism ; Hepacivirus -- Physiology ; Occludin -- Metabolism ; Scavenger Receptors, Class B -- Metabolism ; Tetraspanin 28 -- Metabolism ; Viral Proteins -- Metabolism
    ISSN: 0022538X
    E-ISSN: 1098-5514
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
Close ⊗
This website uses cookies and the analysis tool Matomo. Further information can be found on the KOBV privacy pages