scholarly journals Hepatitis C virus promotes virion secretion through cleavage of the Rab7 adaptor protein RILP

2016 ◽  
Vol 113 (44) ◽  
pp. 12484-12489 ◽  
Author(s):  
Ann L. Wozniak ◽  
Abby Long ◽  
Kellyann N. Jones-Jamtgaard ◽  
Steven A. Weinman
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Time Course ◽  
Rna Virus ◽  
Adaptor Protein ◽  
Virus Production ◽  
Hcv Rna ◽  
Cell Periphery ◽  
Dynein Motor ◽  
Intracellular Virus

Hepatitis C virus (HCV) is an enveloped RNA virus that modifies intracellular trafficking processes. The mechanisms that HCV and other viruses use to modify these events are poorly understood. In this study, we observed that two different RNA viruses, HCV and Sendai, cause inhibition of ras-related protein Rab-7 (Rab7)-dependent endosome–lysosome fusion. In both cases, viral infection causes cleavage of the Rab7 adaptor protein RILP (Rab interacting lysosomal protein), which is responsible for linking Rab7 vesicles to dynein motor complexes. RILP cleavage results in the generation of a cleaved RILP fragment (cRILP) missing the N terminus of the molecule. Although RILP localizes in a perinuclear fashion, cRILP moves to the cell periphery. Both knockdown of RILP and expression of cRILP reproduced the HCV-induced trafficking defect, and restoring full-length RILP reversed the trafficking effects of virus. For the first 3 d after electroporation of HCV RNA, intracellular virus predominates over secreted virus, but the quantity of intracellular virus then rapidly declines as secreted virus dominates. The transition from the intracellular-predominant to the secretion-predominant phenotype corresponds to the time course of cRILP generation. Expressing cRILP directly prevents intracellular virus accumulation at early times without affecting net virus production. The ability of cRILP to promote virus secretion could be prevented by a kinesin inhibitor. HCV thus modifies cellular trafficking by cleaving RILP, which serves to redirect Rab7-containing vesicles to a kinesin-dependent trafficking mode promoting virion secretion. Cleavage of a Rab adaptor protein is thus a mechanism by which viruses modify trafficking patterns of infected cells.

scholarly journals Visualization of Double-Stranded RNA in Cells Supporting Hepatitis C Virus RNA Replication

2007 ◽  
Vol 82 (5) ◽  
pp. 2182-2195 ◽  
Author(s):  
Paul Targett-Adams ◽  
Steeve Boulant ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Three Dimensional ◽  
Rna Replication ◽  
Hcv Rna ◽  
Double Stranded Rna ◽  
Core Proteins ◽  
Virus Rna ◽  
Infected Cells

ABSTRACT The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.

scholarly journals The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle

2020 ◽  
Vol 21 (4) ◽  
pp. 1479 ◽  
Author(s):  
Cristina Romero-López ◽  
Alfredo Berzal-Herranz
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Long Range ◽  
Genomic Sequence ◽  
Rna Virus ◽  
Hcv Rna ◽  
Structural Elements ◽  
Control Translation ◽  
Rna Interaction

RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.

scholarly journals An RNA-Binding Protein, hnRNP A1, and a Scaffold Protein, Septin 6, Facilitate Hepatitis C Virus Replication

2007 ◽  
Vol 81 (8) ◽  
pp. 3852-3865 ◽  
Author(s):  
Chon Saeng Kim ◽  
Su Kyoung Seol ◽  
Ok-Kyu Song ◽  
Ji Hoon Park ◽  
Sung Key Jang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Virus ◽  
Hcv Rna ◽  
Hnrnp A1 ◽  
Host Proteins ◽  
Nontranslated Region ◽  
Septin 6

ABSTRACT Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus. NS5b is an RNA-dependent RNA polymerase that polymerizes the newly synthesized RNA. HCV likely uses host proteins for its replication, similar to other RNA viruses. To identify the cellular factors involved in HCV replication, we searched for cellular proteins that interact with the NS5b protein. HnRNP A1 and septin 6 proteins were identified by coimmunoprecipitation and yeast two-hybrid screening, respectively. Interestingly, septin 6 protein also interacts with hnRNP A1. Moreover, hnRNP A1 interacts with the 5′-nontranslated region (5′ NTR) and the 3′ NTR of HCV RNA containing the cis-acting elements required for replication. Knockdown of hnRNP A1 and overexpression of C-terminally truncated hnRNP A1 reduced HCV replication. In addition, knockdown of septin 6 and overexpression of N-terminally truncated septin 6 inhibited HCV replication. These results indicate that the host proteins hnRNP A1 and septin 6 play important roles in the replication of HCV through RNA-protein and protein-protein interactions.

Chronic Hepatitis C Virus Infection in Haemophilic Patients: Clinical Significance of Viral Genotype

1995 ◽  
Vol 74 (05) ◽  
pp. 1259-1264 ◽  
Author(s):  
P T Telfer ◽  
H Devereux ◽  
K Savage ◽  
F Scott ◽  
A P Dhillon ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clinical Significance ◽  
Time Course ◽  
Infection Type ◽  
Hcv Rna ◽  
Type I ◽  
Hcv Genotype ◽  
Type 3

SummaryWe have undertaken a comprehensive study of hepatitis C virus (HCV) genotype and its clinical significance in haemophilic patients. 189 HCV RNA positive patients were typed, using the Simmonds classification scheme, by restriction fragment length polymorphism (RFLP) in an amplified segment of the 5 non-coding region of the HCV genome. Type 1 was found in 121 (64.0%), type 2 in 23 (12.2%), type 3 in 36 (19.0%), type 4 in 3 (1.6%), type 5 in 2 (1.1%) and mixed infection in 3 (1.6%). There were no type 6 infections and one patient (0.5%) could not be typed. Genotype was not associated with diagnosis, age, or with HIV infection. Type I was associated with higher serum HCV RNA levels, and with a poor response to interferon. Progression to hepatic decompensation has been seen less frequently in those with type 3 compared to type 1 infection (p = 0.07). Three out of eleven patients studied over a longer time course showed a change in genotype, the remainder were persistently infected with HCV type 1. In conclusion, HCV genotype has clinical relevance in the management of haemophilic patients. Those with type 1 are probably more likely to develop serious liver disease and since they respond poorly to inter- feron-α, should be considered for new treatment strategies aimed at sustained clearance of HCV RNA.

scholarly journals Serum hepatitis C virus sequences in posttransfusion non-A, non-B hepatitis

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1157-1160
Author(s):  
M Shibata ◽  
T Morishima ◽  
T Kudo ◽  
T Maki ◽  
S Maki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Chronic Disease ◽  
Hepatitis C ◽  
Time Course ◽  
Clinical Status ◽  
Hcv Infection ◽  
Acute Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hcv Rna ◽  
Negative Case

We investigated 17 patients (12 males and 5 females, ages 2 to 57 years old) with posttransfusion non-A, non-B hepatitis to determine relationships between clinical courses and hepatitis C virus (HCV) markers. The patients were grouped according to time course of abnormal serum alanine aminotransferase (ALT) levels into three categories (chronic biochemical disease, biochemically resolved chronic disease, and acute disease). Latest serum samples (1.3 to 10.8 years after blood transfusion) were used to detect antibodies against C100–3 antigen (anti-HCV) by enzyme-linked immunosorbent assay and HCV sequences by polymerase chain reaction (PCR) assay. Of the 17 patients, 13 patients (76.5%) were anti-HCV positive and 8 patients (47.1%), including one anti-HCV negative case, were positive for HCV RNA. In total, 14 patients (82.4%) were positive for either HCV markers. With respect to clinical course, HCV RNA was detected in six of eight patients (75%) with chronic biochemical disease, and in two of five patients (40%) with biochemically resolved chronic disease. HCV RNA was not detectable in convalescent sera from four patients with acute disease. These results show that there is a relationship between clinical status and HCV viremia, but that normal liver function tests do not always represent the clearance of the virus. Viremia in two patients with normal ALT level suggests that hepatitis is not only caused by viral cytopathic effects, but also by immunologic reactions against virus- infected cells. Thus, PCR is useful in determining the persistence of HCV infection as well as to diagnose anti-HCV negative HCV infection.

scholarly journals Endocannabinoid CB1 antagonists inhibit hepatitis C virus production, providing a novel class of antiviral host-targeting agents

2014 ◽  
Vol 95 (11) ◽  
pp. 2468-2479 ◽  
Author(s):  
Mahsa Shahidi ◽  
Enoch S. E. Tay ◽  
Scott A. Read ◽  
Mehdi Ramezani-Moghadam ◽  
Kazuaki Chayama ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Cannabinoid Receptor ◽  
Virus Production ◽  
Hcv Rna ◽  
Virus Infectivity ◽  
Subgenomic Replicon ◽  
Cb1 Antagonists

Direct-acting antivirals have significantly improved treatment outcomes in chronic hepatitis C (CHC), but side effects, drug resistance and cost mean that better treatments are still needed. Lipid metabolism is closely linked with hepatitis C virus (HCV) replication, and endocannabinoids are major regulators of lipid homeostasis. The cannabinoid 1 (CB1) receptor mediates these effects in the liver. We have previously shown upregulation of CB1 receptors in the livers of patients with CHC, and in a HCV cell-culture model. Here, we investigated whether CB1 blockade inhibited HCV replication. The antiviral effect of a CB1 antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), was examined in HCV strain JFH1 cell-culture and subgenomic replicon models. The effects on the expression of genes involved in lipid metabolism were also measured. CB1 short hairpin RNA (shRNA) was used to confirm that the effects were specific for the cannabinoid receptor. Treatment with AM251 strongly inhibited HCV RNA (~70 %), viral protein (~80 %), the production of new virus particles (~70 %) and virus infectivity (~90 %). As expected, AM251 reduced the expression of pro-lipogenic genes (SREBP-1c, FASN, SCD1 and ACC1) and stimulated genes promoting lipid oxidation (CPT1 and PPARα). This effect was mediated by AMP-activated protein kinase (AMPK). Stable CB1 knockdown of cells infected with HCV showed reduced levels of HCV RNA compared with controls. Thus, reduced CB1 signalling inhibits HCV replication using either pharmacological inhibitors or CB1 shRNA. This may be due, at least in part, to reduced lipogenesis, mediated by AMPK activation. We suggest that CB1 antagonists may represent an entirely new class of drug with activity against HCV.

scholarly journals Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

2001 ◽  
Vol 75 (24) ◽  
pp. 12047-12057 ◽  
Author(s):  
Peter Friebe ◽  
Volker Lohmann ◽  
Nicole Krieger ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Rna Replication ◽  
Hcv Rna ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Nontranslated Region ◽  
High Level ◽  
Hcv Ires

ABSTRACT Sequences in the 5′ and 3′ termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5′ end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5′ NTR required for RNA replication. We show that deletions introduced into the 5′ terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5′ NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5′ NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5′ NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus.

scholarly journals Regulation of Hepatitis C Virus Translation and Infectious Virus Production by the MicroRNA miR-122

2010 ◽  
Vol 84 (13) ◽  
pp. 6615-6625 ◽  
Author(s):  
Rohit K. Jangra ◽  
MinKyung Yi ◽  
Stanley M. Lemon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Virus Production ◽  
Hcv Rna ◽  
Wild Type ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
High Virus ◽  
In Cells

ABSTRACT miR-122 is a liver-specific microRNA that positively regulates hepatitis C virus (HCV) RNA abundance and is essential for production of infectious HCV. Using a genetic approach, we show that its ability to enhance yields of infectious virus is dependent upon two miR-122-binding sites near the 5′ end of the HCV genome, S1 and S2. Viral RNA with base substitutions in both S1 and S2 failed to produce infectious virus in transfected cells, while virus production was rescued to near-wild-type levels in cells supplemented with a complementary miR-122 mutant. A comparison of mutants with substitutions in only one site revealed S1 to be dominant, as an S2 but not S1 mutant produced high virus yields in cells supplemented with wild-type miR-122. Translation of HCV RNA was reduced over 50% by mutations in either S1 or S2 and was partially rescued by transfection of the complementary miR-122 mutant. Unlike the case for virus replication, however, both sites function equally in regulating translation. We conclude that miR-122 promotes replication by binding directly to both sites in the genomic RNA and, at least in part, by stimulating internal ribosome entry site (IRES)-mediated translation. However, a comparison of the replication capacities of the double-binding-site mutant and an IRES mutant with a quantitatively equivalent defect in translation suggests that the decrement in translation associated with loss of miR-122 binding is insufficient to explain the profound defect in virus production by the double mutant. miR-122 is thus likely to act at an additional step in the virus life cycle.

scholarly journals Role of RNA Structures in Genome Terminal Sequences of the Hepatitis C Virus for Replication and Assembly

2009 ◽  
Vol 83 (22) ◽  
pp. 11989-11995 ◽  
Author(s):  
Peter Friebe ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Hcv Rna ◽  
Rna Structures ◽  
Rna Packaging ◽  
Stem Loop ◽  
Nontranslated Region ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Hepatitis C virus (HCV) is a positive-strand RNA virus replicating its genome via a negative-strand [(−)] intermediate. Little is known about replication signals residing in the 3′ end of HCV (−) RNA. Recent studies identified seven stem-loop structures (SL-I′, -IIz′, -IIy′, -IIIa′, -IIIb′, -IIIcdef′, and -IV′) in this region. In the present study, we mapped the minimal region required for RNA replication to SL-I′ and -IIz′, functionally confirmed the SL-IIz′ structure, and identified SL-IIIa′ to -IV′ as auxiliary replication elements. In addition, we show that the 5′ nontranslated region of the genome most likely does not contain cis-acting RNA structures required for RNA packaging into infectious virions.

scholarly journals Amphipathic α-Helix AH2 Is a Major Determinant for the Oligomerization of Hepatitis C Virus Nonstructural Protein 4B

2010 ◽  
Vol 84 (24) ◽  
pp. 12529-12537 ◽  
Author(s):  
Jérôme Gouttenoire ◽  
Philippe Roingeard ◽  
François Penin ◽  
Darius Moradpour
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Major Determinant ◽  
Hcv Rna ◽  
Intact Cells ◽  
Replication Complex ◽  
Α Helix ◽  
Positive Strand Rna

ABSTRACT Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic α-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.

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