Lecture 27 Flashcards
(31 cards)
1
Q
Who are the three people who got the Nobel prize for HCV?
A
- Alter in 60s and 70s started to alert the public that a non A non B hep was prevalent at clinics and hospitals in transfusion patients and people were getting sick became known as transfusion associated or non A non B hep
- Houghton discovered and isolated the virus for the 1st time while working at Chiron
- Rice took virus and infected chimpanzees and showed that it caused hepatitis C virus infetcuon
2
Q
How did Houghton discover Hep C?
A
- 1989 by Houghton who now Canada excellence research chair trying to develop vaccine
- Patients tested negative for Hep A and Hep B
- HCV, etiologic agent of the disease was discovered by Houghton and investigators while working at Chiron, Inc
- Complicated process => extracted RNA from blood/plasma from patients presenting with nonA non B hep, then RT to get cDNA which was digested with EcoRI and put the little pieces into bacteriophages that then infected E. coli => this makes a phage display since they express little pieces of the cDNA, little proteins and the E. coli expresses the proteins and following plating, they would take serum from nonA nonB patients and pour it over the plate and looked for colonies where Ab would stick to the bacteria presenting those peptides, locate each of the colonies that lit up and would clone those and reexpress and sequence the little fragments until the whole genome was put together
Huge tour de force, took many years to complete and one of the reasons why Houghton received the Nobel prize
3
Q
About HCV?
A
- 170 million people used to be infected worldwide (about 3% of population) but now only 71 million thanks to DAAVs (effective drugs), over 286,000 Canadias, nearly 5x the burden of HIV
- Responsible for 40-60% of all chronic liver disease worldwide and it’s the leading cause of liver transplant worldwide
- Of those infected approx 85% became chronically infected, never really clear virus unless receive antiviral therapy
- No vaccine available to date
4
Q
Prevalence of HCV worldwide?
A
- All over the world but higher in Northern Africa, central and south east Asia, lowest in Brazil, Canada and USA and moderate everywhere else
- Higher in Egypt about 14.7% because in the 80s vaccinating agains schistosomiasis (parasite) with no sterile technique, reused needles and gave about 15% of the population HCV when they were children so now a lot of people with chronic HCV are quite young in their 30s, antivirals huge impact in disease burden in Egypt
5
Q
The different viral hepatitis?
A
Viruses causing liver inflammation although very Dif viruses
A: picornaviridae, fecal-oral
B: Hepadnoviridae, DNA virus, blood/sexual
C: flaviviridae family but not insect borne and is hepacivirus genus not flavivirus, blood
D: viroid-like, satellite virus, does not have own packaging proteins, uses Hep B proteins in co-infection (blood)
Less common ones
E: calicyviridae distantly related to picornaviruses, fecal/oral
F: unknown, fecal-oral
G (GB virus C): flaviviridae, blood
6
Q
How is HCV transmitted?
A
- Blood transfusion used to be most common (Canada gave thousand of people HCV bus improper screening of blood and not quickly enough) but now it’s through injection drug use, then high risk sexual activity, not standard STD but if blood products, can be transmitted through sex
- Also through health care workers, hemodialysis, mother to child (low)
- Unknown but likely to be household exposure, accidental injuries, cocaine use cuz sniffing and nosebleeds, tattling, dentistry, unsafe sterile technique
7
Q
How to diagnose?
A
- not a part of standard STD panel must ask doc for screening if you feel at risk
- serology (enzyme immunosorbent assay). look for anti-HCV Ab in blood once exposed to infection although doesn’t indicate with 100% certainty an active infection, in over 95% of chronically infected patients
- Viral genome copies (qPCR) only after positive serology test, look for viral RNA in the blood (active infection), genotyping => cuz lots of Dif genotypes of HCV
- Assess degree of liver damage by looking for serum liver enzyme elevation; fibroscan (liver inflammation and fibrosis) liver ultrasound to determine liver damage/fibrosis, used to do liver biopsies but now thanks to fibroscan, only done when absolutely necessary through neck vein instead of side abdomen jab
8
Q
Consequences of HCV infection (1st step)?
A
Start with acute hepatitis
- Incubation period of 6 to 10 weeks before symptoms
8% with acute HCV will have no symptoms so may not know that HCV positive
- If symptoms, can include: pain in upper right quadrant (where liver is), anorexia, abdominal pain, nausea, vomiting, fever, fatigue, and jaundice (only 25%, quite rare despite popular belief that hepatitis = jaundice)
- Approx 15% will clear the infection, 85% will develop chronic infection, unknown why some clear while other may chronic
9
Q
2nd step of HCV infection?
A
- Chronic infection
- Continuing HCV-related disease without improvement for at least 6 months
- 60-80% have no symptoms so many will not know they’ve been infected until 10-20 years later when going into liver failure, 20 years could elapse between initial infection and development of serious complications
- slowly progressing, lifelong infection
- Cirrhosis (scar tissue) and liver failure (10-20%)
- Hepatocellualr carcinoma (liver cancer, 3-5%)
- Typically clinical symptoms appear during liver failure
- Nodular cirrhosis => accumulation of scar tissue
- CT abdominal transverse => enlarged liver/inflammed and a dark spot is hepatocellular carcinoma
10
Q
What is the natural course of HCV infection?
A
- HepC infection => acute Hep C => 85% chronic while 15% virus no longer detected => 20-25% cirrhosis while the rest minor liver damage => liver failure in 10-20%, Liver cancer in 3-5% and cryoglobulinemia when liver enzymes spilled in blood and precipitate in cold TºC so blood starts to precipitate, this is a rare complication but common for HCV, exact percentage is difficult to calculate because HCV affects more people in warm places
11
Q
HCV family, genus, morphology?
A
Flaviviridae, very distant from other flaviviruses so looks alone in family but the family IS expanding
Hepacivirus
Enveloped, (+)ssRNA about 9.6 kb
12
Q
HCV genome organization?
A
- Single long ORF (about 3000 aa poly protein) cleaved by host and viral proteases into the 10 mature viral proteins
- No cap, has triphosphate end and then IRES to recruit ribosome since no cap but is ribosome ready so translation upon entry and uncoating in the cell, then the structural proteins (C core/capsid, E1 and E2 envelope proteins, p7 possibly an ion channel) then non structural proteins (NS2 auto protease that cleaves boundary between NS2 and NS3, NS3 protease that cleaves the rest of the boundaries in non structural proteins, also is a helicase, 4A protease co-factor for functional protease, NS4B membranous web or replication organelle, NS5A RNA replication and replication organelle biogenesis, NS5B RdRp for viral genome replication) and no poly A tail, just a very structured 3’ UTR
13
Q
Why do we call RNA the workhorse of HCV?
A
- must fold in specific structures to mediate different steps of viral life cycle like pseudo knots and kissing loops
- Involved in translation (IRES)
- RNA replication pseudoknot, kissing loop and SLI
- And packaging
14
Q
How are HCV particles so interesting?
A
-They are lipoviral particles (LVP) named this way due to tight association with HDL/LDL (liver lipoproteins made or found there)
- Either two particle model, interacts with lipoproteins through apo proteins C and E
- Single particle model (virion particle partially enclosed in LDL molecule) and this model seems to be the most accurate
- HCV particles have a low and heterogenous buoyant density for an enveloped RNA virus (less or equal to 1.10 g/mL) thanks to association with lipoproteins and there can be a different amount of lipid per particles
15
Q
How does HCV enter the cell?
A
- Can take advantage if the LDL receptors for entry thanks to tight association
- GAG is first interaction then LDLR (both co-recpetors) since LVP then more specific interaction with SR-BI (co-receptor) and CD81 (receptor) at the surface of the hepatocyte and these two will drag virus particle to tight junction where virus can interact with CLDN1 and OCLN (both receptors) to trigger clathrin mediated endocytosis
- many co-receptors before finally entering the cell in the endosome => release and uncoating and translation can start
16
Q
How does translation work for HCV?
A
- HCV is not capped
- Cap independent translation accomplished by IRES that grabs ribosome
- IRES is made up of SLII to IV of the 5’ NCR and binds to 40S subunit of ribosome uses only eiF2 and 3 then places the start codon on the P site of the ribosome for initiation no need to scan like in polio that has IRES but ribosome needs to scan
17
Q
How is HCV poly protein processed?
A
- Host proteases cleave structural protein boundaries then NS2 does boundary of NS2/NS3 and NS3 does all the other boundaries in non structural proteins
- Every single viral protein has a transmembrane domain except NS3 but it has its cofactors NS4A that has a transmembrane domain, this virus is very highly associated wutg ER membrane even RdRp has transmembrane domain, replication takes place in membranous web
18
Q
What proteins make up the replication complex?
A
- NS3 through NS5B form the replication complex/replication organelle
- NS3 helicase unwinds RNA for replication, NS4A is cofactors
- NS4B membrane reorganization of ER to make replication organelle
- NS5A phosphoprotein, RNA replication and assembly possibly
- NS5B RdRp copies viral RNA
19
Q
What are the famous HCV membranous webs?
A
- In association with lipid droplets
- Common to many positive-strand RNA viruses like polio and flaviviruses although replication organelle differs slightly but all vesicular or membrane associated
- Functions: physical support to have replication and build the replication membrane, compartmentalization of replication, protection of viral RNA because kept away from nucleases and cellular sensors of forming RNA, tethering of RNA for unwinding, retention of the negative strand which will serve for synthesis of lots of (+)ssRNA in one place
- Derived from the ER: lipid constituents (lipid droplets, LDs), DMVs tethered to the ER membrane, MMVs later in infection
- Induced by NS4B and NS5A
20
Q
But then how does the genome escape the membranous webs?
A
- vesicles contain pores (although not known exactly how, may be nuclear pore complex) that allow transport of viral genome RNA in and out
- The pore stains with nuclear pore complex-specific antibodies
- NLS can direct proteins to the inside of vesicles
- Solid evidence of nuclear pore complex sequestering
21
Q
About NS5B, the RdRp?
A
- Right hand topology, initiation de novo, no primer, just starts with 2 NTs together to initiate, GTP is generally accepted as the initiating NTP (in vitro) (could also be A), A/G are bigger NTs that could be need for proper initiation
- Requires divalent metal ions
- Very highly error-prone (replication rate = 10 to the 12 viral particles a day)
- Result: spectrum of genetic variant in every species, so a quasispecies
22
Q
The diversity of HCV?
A
Highly diverse due to error-prone polymerase
- HIV thought to be super divers but BOY HCV way more mistakes than HIV
- Initial spectrum of viruses not just a single virus which affects disease progression and treatment
- Genotype (30-35% difference between genotypes), 10-30% difference between subtypes
23
Q
The HCV assembly puzzle
A
- Many pieces but not exactly known how they fit together
- E1 and E2 definitely involved in assembly and Core condense RNA and NS5A seems to bring RNA to the core protein so there’s a physical connection there too
- NS2 and E2 colocalize
- Genetic interactions between NS2 and NS2, NS3, NS5A, E1
- Genetic interaction between p7 and core and p7 and E1
- Genetic interaction means that the mutation in one could be rescued by mutation in the other
24
Q
How does the virion assemble and exit?
A
- Structural and non structural proteins participate in assembly
- Replication happens in replication organelle then NS5A traffics RNA to assembly so at lipid droplet where the core protein accumulates so thought that assembly initiates ay the surface of the lipid droplet, then the core condensed RNA and interacts with E1/E2 to build virion on the surface and budding into the ER membrane to form nucleocapsid and envelopment happens at the same time so we can’t have nucleocapsid without envelopes in HCV and other proteins involved like p7, NS2, NS3 and NS4A
- HCV can then hijack VLDL pathway probably to exit cell and that’s probably how they end up associated to LDL/HDL in order to be secreted
25
What is the role of p7?
- viroporin or ion channel involved in exit
- promotes pH mediated maturation and release of infectious viral particles from the cell into viral lumen
26
How does HCV max coding capacity?
- Polyprotein and internal initiation
27
How can we study HCV?
- After Houghton baddie
- Rice put HCV in chimpanzees in 1997, first animal model, can only use primates as animal models since HCV doesn't infect mice or other small mammals BUT no longer ethical with chimpanzees unless vaccine research so no more animal model
- Development of drugs and vaccines requires a system to study the virus
- Only recently been able to culture virus in cell cultures 1999 ten years after virus discovery though was possible to extract from patients
28
What are the 3 culture systems for HCV?
- HCVpp
- Replicon
- HCVcc
29
HCVpp?
Pseudoparticles, fake viral particles, have envelope proteins on surface
- Transfect into 293T cells (highly transfectable) LTR-provirus-LTR with reporter like luciferase, HCV E1/E2 and HIV gag-pol, HIV encapsidates the reporter virus and envelope proteins decorate surface and we obtain the pseudo particles which can infect Huh-7 cells (liver cells) that will express luciferase so we looks for reporter expression
- pros: easy to perform, can look for entry inhibitors, glycoprotein mutagenesis
- Cons: particles made in non-hepatic cells so liver specific factors like LDL could be missing from particles, glycosylation is different since absence of liver specific factors, misfolding of E1/E2 due to missing liver specific factors/or other viral proteins that help proper E1/E2 folding, inefficient low yield and decoration of E1/E2 could be heterogenous and may not reflect the true decoration of E1/E2
30
HCV replicon?
- Facilitated study of RNA synthesis (that particular step of viral life cycle)
- 1st model to allow for study of RNA replication
- Took cDNA of genome and cut out structural proteins and replaced with a reporter or selection marker like luciferase or antibiotic and then added another strong viral IRES to drive production of non-structural proteins then took HCV replicon RNAand electroporate to introduce into liver cell line Huh-7 cells and look at transient/stable with antibiotic resistance RNA replication and reporter gene expression
Pros: non infectious can't make particles because no structural proteins is can replicate in cell but can't leave cell, isolates the replication step so study protease, polymerase, and look for NS5A inhibitors and inhibitors of other viral enzymes, multiple genotypes can be studied cuz can make Dif versions of this
- Cons: ignores contribution of structural proteins, results might not translate to infectious cycle bus artificially driving translation from EMCV IRES so lots of things could get and affected and not be in infectious cycle
31
HCVcc?
Infectious HCV only studied in 2004-2003
Akita isolated an HCV sequence from a Japanese patient with fulminant hepatitis JFH-1 severe form of acute hepatitis infection, very symptomatic and high viral load
- Non-structural genes were shown to produce 1000x more viral RNA than that of other HCV isolates from this replicon
- Akita and Rice full length JFH-1 was shown to produce infectious HCV in cell culture and the infectious clone was born => cell culture-derived HCV HCVcc
- Only possible to study full viral life cycle in infection when we got HCVcc
- JFH-1 produces infectious virus in cell culture but still quite low extent (10 to the 2 and 10 to the 3 flu/mL), developed Huh 7.5 cells which are a sub clone of Huh7 with an inactivated mutation in RIG-I RNA sensor and then when cells transfected can get good levels of virus in cells to study
- Chimeric virus now exist in al 7 genotypes (with JFH-1) producing various levels of infectious virus but importantly ALL of them must have the JFH-1 non structural proteins, without them cannot get infectious particle production or very weak phenotype
- Pros: structural genes from all genotypes can be studied
- Cons: completely artificial cuz all contain JFH-1 nonstructural gene and structural proteins of other genotypes
- Very recent so this is why assembly has not been well studied yet
