Hepatitis C Virus Flashcards
Hepatitis C Virus -> General information
Family: Flaviviridae
Genera: Flavivirus, Hepacivirus (Hepatitis C Virus, HCV), Pegivirus, Pestivirus (Bovine viral diarrhea virus 1 (BVDV-1), Classical swine fever virus (CSFV))
Genome: RNA
- single stranded
- positive polarity
- size of 9.5 kb
- IRES element
- no poly-A
Gene expression: Single open reading frame -> Polyprotein
Virions: 40-60 nm in diameter, Lipid envelope
Global prevalence:
- 71 Mio. chronic cases world wide
- 399 000 death 2016
Hepatitis C Virus (HCV) -> Detection and discovery
- Formerly non A / non B hepatitis
- 1987 discovery of HCV genome in patient material (Chiron Corp., CA., USA)
- ELISA for the detection of anti-HCV antibody - Western Blot and RT-PCR detection system
-> No new infections via blood transfusions/blood products!
HCV-Infections in Western Europe
- 5 Mio chron. infected persons
- 20% of all acute hepatitis
- 70% of all chronic hepatitis cases
- 40% of all final stage cirrhosis cases
- 60% of all hepatocellular carcinomas
- 30% of all liver transplantations
- ca. 12.000 deaths each year
Risk factors for HCV-infection
60 % iv drugs
15 % sexual
10 % Transfusion (untested blood products)
5 % Else (Nosocomial, Public Health Service, Perinatal)
10 % Unknown
Sexual and perinatal communication compared to HBV far less relevant
After 20 years of sexual relationship 5 % chance of communication to the partner
Hepatitis C -> behavior
- Communication by blood, blood products, associated risk factors
- Extrahepatic manifestation (mixed cryoglobulinaemia; B-cell anomaly)
- High genomic variability of HCV
- Successive HCV infections possible
(no sufficient cross-immunity) - No vaccine
Hepatitis C: clinical features
- Incubation periode: 2-26 weeks (6-7)
- Immunity: no permanent protection (reinfection)
- Vaccine: not in sight
- Therapy: PEG-interferon + Ribavirin, FDA approved: since 2014 several directly interacting drug
Course of hepatitis C virus infection
25% apparent -> 20% Virus elimination (within 6 months)
75% inapparent -> 80% persistent infection -> ca. 60% chronic hepatitis -> (1-3 decades) -> 10 - 20% liver cirrhosis (ca 40% of LC) -> (2-10 years) -> Hepatocellular carcinoma (ca. 60% of HCC) (1-5% each year)
HCV - pathogenesis
healthy liver -> inflammation; fibrosis -> cirrhosis -> Hepatocellular cancer
Summary of HCV pathogenesis
- HCV frequently establishes persistent infections
- HCV infection causes a complex disease
- liver cell destruction caused mainly by immune response
- contribution of viral cytopathogenicity unclear
- even after successful virus elimination elevated risk for liver cancer
Problem for any study:
- Only valid animal model is chimpanzee (now also mice with human liver cells; see below)
- Consequence: Many data only from cell culture
Course of viral infections
Acute infection -> e.g. influenza virus, Rhinovirus
Chronic infection -> e.g. hepatitis C virus
Latent infection -> e.g. herpes simplex virus (HSV)
Slow infection -> e.g. HIV
Immunological reactions against viral infections
- Innate immune system
- interferone system
- macrophages
- „natural“ killer cells (NK)
-> Period between infection and activation: hours - Cellular immune response
- T-killer cells (CD8)
- T-helper cells (CD4)
-> Period between infection and activation: days - Antibodies
- neutralizing Abs are important for the protection against reinfection
- main determinant for success in vaccination
- does not allways protect against infection with identical pathogens e.g. influenza virus, hepatitis C virus, HI virus
-> Period between infection and activation: ca 1-2 weeks
HCV: Strategies of virus persistence
1. Strategy: suppression of the innate immune system
- NS3 needs NS4A as cofactor for full protease activity; is essential for viral replication
- NS3/4A complex cleaves TRIF:
- no signaling through TLR 3 (dsRNA)
- no signaling through TLR 4 (viral glycolipids)
- NS3/4A complex cleaves Cardif (= MAVS; VISA; IPS-1)
- no signaling through RIG-I or Mda-5
- no recognition of cytoplasmatic dsRNA /RNA with 5 ́triphosphate
HCV: Strategies of virus persistence
2. Strategy: very high antigenic variability
7 genotypes (8th?): - 30 -35% divergence on RNA level
- within a single genotype 20 – 25% difference (RNA)
Consequence:
- often no cross-neutralisation
- several vaccines needed
Estimated number of formed viruses/per patient and day: ca. 1011-1012 (half live of free virus 11-19 h)
Variance per genome copy: between 1 and 10 exchanges
- single treatment with Telaprevir® eliminates 99.97% of virus in patient
- still 107 viruses per patient replicating
- rebound of titer within 7 – 10 days
Preexisting resistant mutants are a major problem
- footrace: Antigenic main species is attacked via clonal propagation of B- and T-cells with matching antigen receptors
- minor representatives of quasispecies are not eliminated and thus spread
Antigenic variability is critical for the entire adaptive immune response
HCV: different genotypes in different countries
USA; Canada: 1a, 1b, 2a, 2b, 3a
South America: 1a, 1b, 2, 3a
Europe: 1a, 1b, 3a
Northern and Central Africa: 4
South Africa: 5a, 2, 3
Japan, Taiwan, China: 1b, 2a, 2b
Vietnam: 6, 1b, 2
Hongkong: 6a, 1b, 2a, 2b
History of Hepatitis C Virus Research
- until 1989 HAV known, HBV known but high numbers of hepatitis patients without detectable HAV or HBV infection. Terminology: nonA nonB hepatitis
- 1989: Research team of Dr. M. Houghton at Chiron Pharma cloning first piece of HCV genome as cDNA
- cloning of entire genome
- test systems for screening of blood donors (core ELISA, PCR)
But - no cell culture system for HCV isolates from patients (still today)
- no replication of viral RNA in cell culture until 1999
- 1999: Breakthrough! Small lab in Mainz develops HCV RNA replicon system
Basis for all drugs currently in clinical testing against HCV!
Breakthrough: HCV replicon system
Trick:
- reduction of genome size to minimal set of genes required for RNA replication
- integration of a selectable marker (drug resistance gene)
- propagation transfected hepatoma cells (Huh7) under selection pressure: cells without HCV replication die; only cells with replicating HCV RNA express resistance gene and survive
- surviving cells form colonies
- replicating HCV RNA can be isolated, sequenced and compared to input RNA
- cell culture adaptive mutations have to be cloned back into original replicon cDNA
- RNA replicons dervied thereof can replicate their RNA in Huh7 cells
Result: Adaptive mutations allow HCV RNA replication in cultured cells
Replicon system: Basis for all drugs currently tested against HCV!
The molecular basis for cell culture adaption of HCV
HCV WT requires low levels of PI4KA and adapts to high PI4KA expression levels in Huh7-derived hepatoma cell lines.
Model of the role of PI4KA in HCV cell-culture adaptation. Bottom left: PI4KA levels are low in vivo (normal human hepatocytes).
HCV has evolved a mechanism to activate PI4KA via NS5A (blue) and NS5B (green) to generate a PI4P-enriched microenvironment (red hexagons) essential for RNA replication.
Top right: in permissive Huh7 cells, PI4KA levels are increased. Following stimulation, massive amounts of PI4P are generated, which is harmful for most HCV WT isolates.
Bottom right: HCV adapts to increased PI4KA levels by inactivating the PI4KA-stimulating function within NS5A or NS5B via adaptive mutations.
Cell culture adaption in NS5A and NS5B
- In normal human hepatocytes HCV needs to activate the intrinsic low level of PI4Kinase III alpha (PI4KA) molecules by NS5A and NS5B; activated PI4KA recruits cholesterol and glcosphingolipids via OSBP and FAPP2, respectively. This leads to a permissive PI4 phosphate-enriched membrane microenvironment required for
RNA replication. - In hepatoma cells (Huh7) PI4KA levels are massively higher;
in this setting the activation of PI4KA by wt HCV NS5A and 5B blocks RNA replication (delicate balance!) - Cell culture adaptation in Huh7 hepatoma cells requires mutations in NS5A and NS5B to allow for high level RNA replication in the presence of high levels of PI4KA
- In hepatoma cells wt HCV (without adaptive mutations) can only replicate when PI4KA and CKIa are blocked by chemical inhibitors (these inhibitors are a functional substitute for the adaptive mutations in NS5A and 5B)
History of Hepatitis C Virus Research
1989: first HCV cDNA sequence identified from Hep nonA-nonB patient material
1999: Breakthrough! Bartenschlager Lab. in Mainz develops HCV replicon system But still no formation of infectious HCV particles in cell culture!
Observation:
- cDNA derived HCV clones with original sequence are infectious in chimp
- cDNA derived HCV clones with cell culture adaptive mutations are not!
Strong negative effect of cell culture adaptation on virion production
Similarly:
Secretion of HCV core from transfected cells is suppressed by cell culture adaptive mutations
2003
Takaji Wakita lab: First HCV replicon which replicates without cell culture adaptation
Japanese fulminant hepatitis (JFH) strain (strong RNA replicase!)
Insertion of viral structural genes into replicon allows entire replication cycle in cell culture
This strain (+ second one)! No system for standard patient virus but JFH chimeras with structural proteins of all different genotypes!
Two major breakthroughs
- HCV replicon system
- JFH infectious virus
HCV entry: receptor-mediated endocytosis
Receptors:
CD81
Low density lipoprotein (LDL) receptor Scavenger (SR-BI) receptor
Claudin1 (CLDN1)
Occludin (OCDN)
HCV replicative cycle
Entry: receptor mediated endocytosis
Protein translation, processing, RNA replication (in cytoplasm/ at ER membrane)
Membrane topology of HCV proteins
- All proteins are directly or indirectly linked to the membrane
- Membrane topology is critical for RNA replication and processing by cellular ER localized proteases
- Viral proteins are located not only to the membraneus web / ER, but also at mitochondrial membranes (important for the manipulation of host functions and pathogenesis; virion formation?)
NS5A: Membrane incorporation by amphipathic helices -> only into the outer sheet of the bilayers
Model: Oligomers of viral membrane proteins induce membrane bending
Upon release of viral genomic RNA into the cytoplasm of the infected cell, the viral genome is translated into a poly protein that carries the structural and non-structural proteins. The viral non-structural protein NS4B induces the formation of membrane alterations, which serve as a scaffold for the assembly of the viral replication complex (RC). The RC consists of viral non-structural proteins, viral RNA and host cell factors. Within the induced vesicles, viral RNA is amplified via a negative-strand RNA intermediate.
HCV-induced membrane remodeling
Expression of NS3-5A is sufficient to induce formation of Membrane web at the ER
Location of viral RNA replication
Virion morphogenesis:
Infectious virions shown very low density in preparative density gradients!
Why?
Association with apoB apo- lipoprotein B (apoB) and triglyceride rich lipoproteins (TRL)
lipo-viro-particles (LVP)
Architecture of membrane rearrangements induced 16 h after HCV infection
- Membranes are derived from the ER, like in the case of Dengue virus
- However, not by invagination; vesicles are protrusions of the ER membrane (in Dengue infected cells vesicles are invaginations not protrusions of ER)
- More similarity between HCV, Coronaviruses, Nidoviruses and Picornaviruses
- Main components of the membraneous web are single and double membrane vesicles (DMV)
- DMVs are the predominant form
- The vesicles are frequently connected to the ER membrane via a neck-like structure
Model of the predicted role of the Core-coated lipid droplet in the production of infectious HCV
The association of the core-coated lipid droplet with NPC-and RC-rich ER may enhance the interaction of HCV and VLDL.
VLDL is generated frequently and enhanced in the microenvironment where lipid droplets associate with the ER.
This increased concentration may increase the frequency of the association of HCV and VLDL. HCV/VLDL is released as an infectious particle with a low density, whereas HCV particles that are not associated with VLDL are secreted into the culture medium as noninfectious, dense particles.
However, this model does not discriminate the possibility that noninfectious virus particle also associates with or is integrated with some lipoprotein like structure.
HCV: Virion morphogenesis at ER/Lipid droplets interface
- Lipid droplets are loaded with viral and cellular proteins
- Viral proteins at LD membrane mediate interaction between LDs and ER membrane
- Unloading of viral proteins from LDs to generate virions
„Replication organelles“
- membrane spherulae/memb. web
- sites of RNA synthesis
- transfer of genomic RNA to core
Loading of virion with ApoE
- „Lipo-viro-particle“
- Budding into ER, secretory pathway
HCV exploits liver specific miRNA122
- miRNA122 is preferentially expressed in the liver
- miRNA122 regulates fatty acid and cholesterol synthesis - central to liver function
- HCV RNA replication depends on cellular miRNA-122
- Direct interaction of miR-122 with two target sites in the 5‘UTR of the HCV genome (third interaction site in 3`UTR)
- Binding of miR-122 to the 5‘UTR increases the stability and translation of HCV RNA
Liver-specific expression of miRNA122 may contribute to the tissue tropism of HCV
Silencing of miR-122 as an anti-HCV therapy?
HCV therapy with antisense miR-122 could have two-fold effect:
1. Sequestration of miRNA122 would reduce HCV RNA abundance
2. Would affect lipid metabolism in the liver by reducing host steatosis
LNA technology: short modified RNA molecule
-> ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon
Benefits of the LNA technology:
- Increases the thermal stability of duplexes
- Resistant to exo- and endonucleases resulting in high stability in vivo and in vitroapplications
Therapeutic silencing of MicroRNA-122 in Primates with Chronic Hepatitis C Virus Infection
- treatment of chronically infected chimpanzees with a locked nucleic
acid (LNA)–modified oligonucleotide (SPC3649) complementary to miR-122
Findings:
* leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals.
* transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology.
Reduced concentrations of cholesterol and low-density lipoprotein in the high dosis group
But: viral RNA concentrations increased again in serum and liver after the therapy
The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.
miR-122 is an attractive target for antiviral HCV therapy!!
Factors that determine the host cell tropism of HCV
Approach: introduce genes for known host factors into non-permissive cell line 293 T
- receptor
- micro RNA
- lipid metabolism
Therapy against hepatitis C -> Importance of HCV genotype on the success of IFN-alpha therapy (in %)
IFN alpha (genotype 1 -> 6-11 / genotype 2/3 -> 28-37)
IFN alpha + Ribavirin (genotype 1 -> 28-31 / genotype 2/3 -> 64-69)
peg IFN alpha (genotype 1 -> 14-28 / genotype 2/3 -> 47-56)
peg IFN alpha + Ribavirin (genotype 1 -> 40 (65% by addition of Telaprevir - but side effects, 4 x more dropouts during therapy) / genotype 2/3 -> 80)
-> Still a big problem until 2014
-> Medical costs ca 15 000 € per patient (without Telaprevir!); 400.000 pat in Germany! Costs including protease inhibitors about 60 000 €
Therapy against HCV
- 60% of the patients with HCV genotype 1 can not be cured by the pegINF and Ribavirin therapy
Reasons:
- Genotype of virus is critical; to date no association to specific virus genes; to date all cloned virus isolates can be inhibited with IFN in vitro
- Genetics of the host: Different response in Caucasians and others Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance
upstream region of IL28B gene = IFN-l3 = IFN class 3 molecule
Now in the clinic:
- Inhibitors of viral serine protease NS3/4A (first two approved)
- Inhibitors of RNA Pol (NS5B)
- NS5A antagonist (very potent; mechanism unclear)