Module 3.2: Viral Hepatitis Flashcards
Summarise the different families the hepatitis viruses belong to
Hep A Picornavirus Hep B Hepadnavirus Hep C Flavivirus Hep D Deltavirus Hep E Hepevirus
Summarise the different genomes types the hepatitis viruses have
A–>E
ssRNA dsDNA ssRNA ssRNA (viroid-like) ssRNA
Summarise the transition routes of the different hepatitis viruses (A–>E)
Water/poor sanitation Blood Blood Blood Water/poor sanitation
Summarise the disease course of different hepatitis viruses
Acute Acute/Chronic Acute/Chronic Chronic Acute/Epidemic
Summarise the incubation periods of the different hepatitis viruses
30-120 d 45-160 d 15-150 d 30-60 d 15-60 d
Describe the geographic distribution of HepA
More commonly found in Sub-Saharan Africa, East Asia and South America
In these “high epidemic areas” children become infected at a much younger age –> leading to fewer health consequences
Those from “low epidemic areas” e.g. UK, Europe, North America, Australia are usually infected at a later age in life (adults) leading to greater sequalae/consequences
summarise the symptoms of HepA
- Fatigue
- Nausea
- Poor appetite
- Stomach pain
- Mild fever
- Jaundice
Summarise the transmission of HepA
Mainly by the faecal-oral route:
o Contamination of food or water
o Poor hand washing – faecal residues are then transferred to the food.
o Sewage –> Shellfish
• Close person-to-person contact
• Sexual, oral-anal contact
• Less commonly:
In body fluids e.g. blood and saliva [this is more common in Hepatitis B and C]
- Extensive shedding of virus in faeces during 3-6w incubation period and early days of illness
- Causes high prevalence when low levels of hygiene are present
- HAV is very stable at ambient temperature and low pH increasing its longevity in contaminated food and water
- Resistant to acid and detergents – can pass through stomach on entry and leave via the biliary tract on exit
- Secreted from hepatocytes through biliary system into intestine –> faeces
Describe the pathogenesis of HepA
- Viraemia and faecal shedding during the 3-6w incubation
- Gradual increase in viral replication, which correlates with the degree of histopathology
- Associated rise in ALT
- Normal acute response, with the initial IgM response (which falls), followed by sustained IgG response
Majority of those infected recover well and have “life-long immunity”.
Describe the viral genome of HepA virus
SEE p 24 for drawings!!!!
One open reading frame (ORF), encoding a large polyprotein formed of 7500 nucleotides
All structural (form viral capsid) and non-structural proteins (form RNA replication machinery) are covalently linked
• 5’ end
Contains an internal ribosome entry site (IRES)
–> Allows for cap-independent translation via the attachment of ribose polymerase
At the very end of the 5’ end, there is a small peptide (CPG), which allows the polymerase to get inside the viral capsid
• 3’ end
Poly A sequence (tail)
Translation leads to both structural and non-structural proteins
o VP1-3 (and 4, which is attached to 2) form the viral capsid
o The non-coding regions code for proteins that form the RNA replicase machinery
Describe the HepA virus particle
The capsid structure is created by VP1-3 + VP4 (internal
There are 60 copies of each
Form an icosahedral structure
Initially, VP4 remains bound to VP2 when the capsid structure is being formed
o This gives the capsid a porous property allows for the viral RNA to enter the capsid
Once the RNA enters the capsid, the final proteolysis between VP4 and VP2 occurs
VP4 allows for the modification of the shell to lock the RNA within the capsid
Summarise the process of HepA viral invasion and replication
a) Virus entry as HAV-IgA complexes
b) Uncoating and genome release
c) Cap-independent translation
d) Post-translational proteolytic by viral protease, 3Cpro
e) Negative RNA synthesis
f) Positive RNA synthesis
g) Some newly synthesised positive-sense RNA is recycled for further RNA synthesis or translation (dashed lines)
h) Positive-strand RNA molecules packaged into new viral particles
i) Newly assembled HAV particles are secreted across the apical membrane of the hepatocyte into the biliary canaliculus and are then passed into bile and small intestine
Summarise the cell-entry process in HepA
• HAV crosses the intestinal epithelial cells as intact virions to reach hepatocytes
• HAV associated with IgA along the way
• The virus-immune complex is translocated from the apical to the basolateral compartment of polarised epithelial cells via the polymeric immunoglobulin receptor by IgA-mediated reverse transcytosis
• The complex crosses the space of Disse, which is an endothelial layer between endocytes and a sinusoid
- HAV-IgA complexes are infectious for hepatocytes
- HAV binds to TIM1 (T-cell immunoglobulin and mucin domain 1) on the surface of hepatocytes
Summarise the replication process in HepA
• The capsid releases the viral RNA into the cytoplasm, where the replication begins
• 3C protease is the main viral protease for the conversion of polyprotein to its individual proteins
• From the viral strand, the negative RNA is synthesised, which then is synthesised back to the positive RNA
o There are 10x as many genomes (positive-sense RNA) as anti-genomes (complementary/negative-sense RNA)
• The positive strand RNA are packaged into new viral particles and secreted across the apical membrane into the biliary canaliculus
Descibe the hijacking of cell membranes in hepA infection
- HAV released from liver cell apical membranes is unenveloped
- Unenveloped HAV travels to the gut via the biliary system and can be isolated from the “faecal-oral route”
- HAV released from liver cell basolateral membranes is cloaked in host-derived membranes, enveloped viruses (eHAV)
- eHAV is fully infectious and circulates the blood of infected humans
- eHAV formation is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT)
- eHAV can escape from neutralizing antibodies
- eHAV promotes virus spread within the liver i.e. between hepatocyte cells
allows for the virus to have a more rapid production and replication to other hepatocytes
enhanced evolutionary method
Zongdi et al 2013
Describe the host response to HepA
• The viral damage is mainly immunopathic
• Not cytopathic in cell culture and in vivo
• HAV blocks TLR3 and RIG-I signals reducing type 1 IFN protection
• Cytotoxic CD8+ T cells recovered from liver
o They secrete IFN-y stimulates recruitment of inflammatory cells to site of HAV replication
• Anti-HAV IgM produced initially then protective IgG – very rapid response
• It is proposed that activation of this IFN-gamma response is due to eHAV circulation:
o eHAV is released into the blood circulation through the hepatocyte basolateral membrane
o eHAV interacts with pDC cell (in blood) and so results in a Type 1 IFN response
o Type 1 IFN stimulated development of adaptive immune response (hence lymphocyte invasion in liver)
o Naked HAV virions released from apical membrane into biliary ducts gut faeces
Describe how HepA can be prevented
- Improvement in water supply, sanitation and hygiene
- Isolation of infected individuals
- Passive immunisation (IgG)
- Active immunisation (vaccination) best method
Describe the vaccine for HepA
INACTIVATED VACCINE
• Killed virus – incapable of causing active infection
• Killed virus – possesses the antigens which stimulate the production of anti-HAV
• Developed using proven technology (polio, Salk) production process (right)
• Vaccine schedule:
o 3 courses
1st course – at 0 month
2nd course – from 2w-1m
Booster – 6m-12m
• Highly effective – seroconversion rate 99.9-100%
Describe the epidemiology of HepE
- HEV can be an epidemic or sporadic virus
- Mainly in under developed countries
- Zoonotic (i.e. Animal reservoirs) or human to human transmission
- Mortality greater than HAV but may not be age dependent
- Mortality in pregnant women – 20-30%
Describe the pathogenesis of HepE
- Viraemia and fecal shedding occur before symptoms develop greater chance of infection
- IgM and IgG responses are similar to other acute infections, ALT and liver changes are also similar to HAV
- However, the IgG response is not sustained
- The individual may become susceptible again later on in life – problem with vaccine too
Describ the HepE genome
• Genomic RNA 7.2kb in length
• 5’ end
o Contains a cap = detected as a host cell product
o Translation occurs as it would a normal host mRNA
• Positive sense (can be directly translated)
• Contains 3 open reading frames (ORFs)
• Only ORF1 is translated from genomic RNA
o The translation does not go all the way – continues until TAG (STOP codon)
o ORFs 2 and 3 are translated from sub genomic mRNAs
o ORF 1 = polypeptide formed of non-structural proteins involved in making RNA replicase machinery
Describe the role of HEV ORF2 and ORF3 in translation and Processing
• The RNA replicase may bind to the 3’ end of the viral gene and synthesise the anti-genomic RNA in the cellular cytoplasm
• The anti-genomic RNA may undergo two different pathways:
o The RNA replicase may bind on the 3’ end to synthesise the positive viral RNA strand again
o OR the subgenomic RNA may be translated a different positions to synthesise either ORF2 or ORF 3
- ORF2 forms the capsid protein
- ORF3 forms viral release structure i.e. involved in exit of pathogen from hepatocyte
Describe the HepE viral particle
There are 60 copies of the ORF2 protein – which is the minimum size of a viral capsid icosahedral structure
Describe the HepE replication process
similar to HepA
a) Virus binds to receptors (currently receptors have not been characterised proposed HSPGs? HSC70?)
b) Entry into cytoplasm and release of genomic RNA
c) Translation of ORF1 to form RNA replicase
d) Antigenomic RNA synthesis
e) Genomic RNA synthesis and subgenomic RNA synthesis
f) ORF 2 & 3 translation
g) Virus assembly
h) Virus assembly
i) Release of new viruses from hepatocyte (via endoplasmic reticulum)
Describe the Model for Biogenesis of Quasi-enveloped HEV (eHEV)
Yin X et al, Viruses, 2016
- HEV with ORF3 on capsid
- Docking with ESCRT (endosomal sorting complexes required for transport) proteins
- Formation of eHEV in MVB (multivesicular bodies)
- Transport of eHEV in MVB to plasma membrane
- Release of eHEV, resistant to neutralisation by anti-ORF2 antibodies
Summary: thought to be a more efficient way for the virus to transmit to other hepatocytes in different sites of liver.
Note: although most of this virus is released via the basolateral membrane, some is similarly released into the biliary tract like HAV small intestine faeces.
Describe the pevention and Control Measures for Travellers of HEV-Endemic Regions
- Avoid drinking water (and beverages with ice) of unknown purity uncooked shellfish and uncooked fruit/vegetables not peeled or prepared by traveller
- Immunoglobulin prepared from donors in developed countries does not prevent infection
- Unknown efficacy of Ig prepared from donors in endemic areas
- Vaccine (?) –> trialled in China but not successful (possibly due to IgG responses?)
Describe the clinical features of hepatitis B viruses
• Incubation period is 2-3 months
• Leading to clinical illness in a proportion of individuals
o <5 years of age: 10%
o >5 years of age: 30-50%
• Acute case-fatality rate: 0.5-1%
• Chronic infection
o Age<5 years: 30-90%
Depending on the age of the individual the virus can become chronic more likely to occur if you are infected before the age of 5 due to immature immune system (in neonates it can especially be recognised as being a part of the “self” hence reduced response from host immune system)
o Age>5 years: 2-10%
• Mortality from chronic liver disease: 15-25%
o Mortality is dependent on the length of infection
o A lot of morbidity associated with chronic Hep B infection
Describe the epidemiology of HepB
- Around 250 million chronic infections
- 10-20% develop cirrhosis
- Can cause primary liver cancer
- Highest prevalence in Subsaharan Africa and South Asia with fewer but significant number of chronic cases in developed world
Describe liver disease seen in HepB
• Damage is immunopathic
o Host immune response destroys infected liver cells
o Cytotoxic T-cells kill infected cells
o Loss of liver function
o Hepatocytes regenerate from liver stem cells
o Chronic infection hepatocyte replaced by scar tissue – cirrhosis
• Types of infection (age>5)
o Acute = 90%
5-6m duration
Self-limiting (90%)
Fulminant (rare) – very extensive damage
o Chronic = 10%
Quiescent
Active --> cirrhosis
Describe the disease pathogenesis in acute HBV infection
SEE GRAPH p52
• After 1-3m incubation, the HBsAg rises in concentration, with subsequent rise in HBeAg
sustained immunologic response to
- Anti-HBs
- Anti-HBe
- Anti-HBc
• The concentrations of both the surface and e antigens fall within about 6m
Describe the disease pathogenesis in chronic HBV infection
SEE GRAPH p52
• Again, 1-3m incubation period
- Acute viraemia happens in a similar way, with the rise of the surface and e antigens in the serum
- However, there is no response to the surface antigen
- The e antigen may also reside for many years, and eventually are cleared by a delayed antibody response
- Overall failure of immune response to sufficiently clear the infection
- Chronic infection leads to cirrhosis, and possibly HCC
Describ the viral structure of HepB
Outside surface layer
o Three surface proteins – large, medium, small
o Small surface protein is the most abundant, accounts for the viral structure
o The large surface protein is the least abundant, accounts for receptor binding
Inside there is the icosahedral capsid, comprising of the core protein which encapsulates the viral DNA with a polymerase attached to DNA
o 180 copies of core protein
o 27nm nucleocapsid
Describe the HepB genome
• Partial double stranded DNA structure + viral polymerase protein
• HBV genome has a partial missing strand
o Quite a small DNA – 3200 nucleotides (one of the smallest viruses infecting humans)
o 4 open reading frames, which can encode 4 separate protein structures
o Partial overlap of the open reading frames – which allows multiple protein synthesis from same information
LEARN HOW TO DRAW THIS. p53
• Core sequence
o Core + pre-core
o Pre-core is the signal sequence for the production of e antigen
• Surface sequence
o Pre-S1 + Pre-S2 + S
o Translation of just S = small antigen
o Translation of Pre-S2 + S = medium antigen
o Translation of Pre-S1 + Pre-S2 + S = large antigen
• Polymerase sequence
o Overlaps surface sequence completely, and partially overlaps both core and X sequences
o This means that the information is compact to encode for different proteins
o This is an evolutionary advantage, however, it constrains potential mutation ability of the surface antigen as it would ALSO affect the polymerase protein, which is essential for HBV function
Describe the surface antigen proteins in HepB
- Small, medium and large antigens all have similar intermembrane structures (4 units)
- Medium antigen has a longer NH2 terminus than small
- Large antigen has an even longer terminus with MYR (myristoyl group) bound lipid group that allows the virus to interact with hepatocytes and is involved in its stabilisation (along with small and medium antigens)
• HBV also produces aggregates of surface proteins called empty non-infectious subviral particles (SVP): spheres and tubules
o These are the surface envelopes floating around within the infected cell that do not contain HBV itself
o 100-100,000X more present than HBV virion
o Acts as a decoy mechanism immune system is “tricked” into attacking these, hence virus survives for a longer period of time
Describe the HBV core protein
- The core gene features two potential start codes
- If translated from first start code
o The pre-core/core protein produced
o This contains a signal sequence (hydrophobic region) which is secreted into the lumen of the ER
o In the ER, they encounter proteases, which remove the signal sequences + binding domains for nucleic acids this produces e-antigen
o The e-antigen is secreted into the circulation
o Again, the e-antigens have no part in the viral lifecycle, and only act as a down regulatory mechanism against immune response by acting as another decoy protein to inhibit immune response
• If translated from second start codon (ribosomes initiate translation from here)
o Only the core protein without pre-core is produced
o The core protein spontaneously forms the viral capsid, forming hexon and penton 3D structures
SEE FIGURE
Explain the structure of the HBV capsid
- Begins as core protein monomers that dimerise, and eventually form an icosahedral structure
- However, unlike other hepatitis viruses, although it has icosahedral geometry, it has projections out of its main icosahedral structure (interact with lipid bilayer in final outer structure of the virus and also with cytoplasmic domains of surface proteins) and pores to allow molecules through (remember it has reverse transcription occurring in replication cycle, and so it has to be able to allow material such as nucleotides for conversion from RNA precursor to DNA)
Describe the HepB Replication cycle
- HBV is a non-retrovirus that still uses reverse transcription in their replication
- The virus gains entry into the cell by binding to NTCP on the surface –> endocytosed
o Peptide corresponding to HBs-L protein
o Cross-linked to cell receptor
o Purified and characterised
o NTCP (Sodium taurocholate cotransporting polypeptide)
o Functional receptor for HBV BUT it is not efficient so it is proposed that there are likely other receptors involved in viral entry
• The viral genomic DNA is transferred into the nucleus by host proteins called importins (this does not allow capsid to enter the nuclear pore)
• The partially double stranded viral DNA is made fully double stranded by the host repair mechanism
• This is then transformed into supercoiled covalently closed circular DNA (cccDNA) that serves as a template for transcription of the four viral mRNAs
o Structure of cccDNA (right) is very similar to chromosome/cellular DNA so there is no host response to this
• This DNA is then transcribed because it looks like host chromosome material
• This is then translated by RNA Polymerase II in the cytoplasm to form the viral proteins (capsid and surface proteins)
• An mRNA is reverse transcribed to form extra viral DNA copies
SEE FIGURE p54
Describe the different HBV transcripts
• Core
o 3.5kb (greater than genome length)
o Translation core, e-antigen, polymerase proteins
o Template for reverse transcription for the replication of viral DNA
• S1
o 2.4kb
o Translation Pre-S1 (large S)
• S2
o 2.1kb RNA
o Translation Pre-S2 (medium) + S (small)
• X
o 0.7kb RNA
o Translation X protein
Describe the model for HBV polymerase
• The polymerase structure is encoded by 4 regions
o Terminal domain – interacts with various viral nucleic acid structures (binds to stem-loop)
o Spacer – holds domains together (allows re-orientation of TP and RT domains)
o Reverse transcriptase – performs reverse transcription and DNA polymerase actvities
o RNAseH – ribonuclease H domain degrades RNA in dsDNA-RNA hybrid
• Heat shock proteins (HSP70 and HSP40) have been found to bind and remodel domains during priming interactions with stem-loop HBV –> i.e. they have been found to restructure polymerase at different stages of viral replication
• The HBV pregenomic RNA is required for the viral DNA replication
• HBV reverse transcriptase binds to the stem-loop recognition site near the 5’ end of the pregenome
• The stem-loop structure offers a landing platform for the polymerase
• It initiates reverse transcription to the 5’ end
• It then detaches and is able to rebind onto the second stem-loop, again reverse transcribing
• While the DNA is being produced from the polymerase, the RNA is nearly entirely degraded
• Process: referred to as primer strand switching
• This process forms the first (-ve) strand of the HBV DNA
• The second replication is for the +ve strand
o From the –ve strand formed, the positive DNA primer attach and are primed
o The negative strand loops on itself to create a circular sequence
o There is even a small gap the new strand can jump over
o But the polymerase is able to jump over this gap and is able to produce a partially double-stranded molecule
Why it goes around and doesn’t produce a second fully double-stranded DNA molecule is unclear but suggested that this occurs due to the constraint in space inside the viral capsid which only allows for a partially double-stranded DNA molecule
Describe the role of the HBx protein
- Has a transactivating activity – a variety of promoters for viral replication
- Several biochemical activities – but not DNA binding
- Binds p53
- Interferes with DNA repair
- Blocks anti-viral action of cellular restriction factor Smc5/6
Describe the structure of the HBx protein
• Dimerisation region
• UVDDB1 binding
o Affects DNA repair
• P53 binding
SEE FIGURE p56
Describe the background of HepD
Uneven distribution
Common in some parts of America, Eastern Europe and Middle East
SIMULTANEOUS INFECTION of HBV and HDV —> strong host response, eventual loss of both, good prognosis
SUPERINFECTION –> induction of HDV into HBV +ve agent –> sustained presence of viran DNA –> chronic infection in 60-70% - causes severe acute hepatitis, HCC can occur
Describe the structure of HDV
Outer envelope consists of HBsAg from helper virus
Nucleocaspid contains 70 copies of HDAg
Encloses RNA genome
single stranded circular, -ve polarity
Forms a rod like structure due to internal base pairing
Smallest genome of all known human pathogens
Transcription and replication of HDV
HDV receptor on human hepatocyte UNIDENTIFIED but thought to be same as HBV
RNA transferred to nucleus following uncoating
Replication of the genome and synthesis of mRNA carried out by host RNA polymerase II (this is because the genomic and anti-genomic RNA mimics dsDNA) (one study implicated RNA pol I and III)
Genomic RNA used as the template for production of
o An mRNA that is translated into HDAg
o An antigenomic RNA that is full-length, and is produced from greater-than-full length transcripts
• The antigenomic RNA is also covalently circular and single stranded
• Three key features of the genome necessary for transcription
o Origin for the start of RNA synthesis [1639]
o Polyadenylation site downstream of ORF encoding HDAg
o Self-cleavage site capable of self-ligation [ribozyme] cleaves RNA itself
Self-cleaving RNA molecules
Cleave the RNA at specific sites
Present in both genomic and antigenomic strands
Central to the rolling circle mechanism of replication
Linear multimeric RNA is processed by autocatalytic cleavage to yield unit-length RNAs
These are ligated to form circles, by a host RNA ligase or by reversing the ribozyme cleavage reaction
HDV ribozymes are the fastest known self-cleaving RNAs
- So HBV requires the HBsAg coat in order to enter the cell and utilise the cellular machinery
- The L-HDAg is the one that interacts with the HBsAg protein
Functional Domains of HDSAg
nucleocapsid consists of two polypeptides – a small and large surface antigen
o The large one has a farnesylation site – allows binding to lipids (+19aa of small) + suppression of RNA replication
o Small surface antigen is required for replication
Production of L-HDAg
• The large antigen is produced from the antigenome
o The mRNA is modified so that the STOP codon is changed (UAG UGG)
o This change is performed by the host enzyme adenosine deaminase 1 (ADAR1) – where deamination occurs
o One in three mRNA are changed by the enzyme
• L-HDAg is produced later in the infectious cycle
o It suppresses RNA replication and promotes encapsidation
o An isoprenylation signal from the L-HDAg is required for interaction between HBsAg
o HDV capsids contain both HDAgs, and bud off the ER membrane picking up their HBsAg envelope in the process
Summarise the replication cycle of HDV
- The virion attaches to the hepatocyte via an interaction between large-HBsAg and an uncharacterised membrane receptor in the host cell;
- the virion enters the cell and is uncoated;
- the RNP is targeted to the nucleus;
- genomic RNA is transcribed in the nucleus to form antigenomic RNA, which forms the template for replication of new transcripts of the circular genome, and mRNA, which contains the open reading frame;
- the mRNA is exported to the cytoplasm where it is translated at the endoplasmic reticulum to form new molecules of hepatitis D antigen;
- the new antigen molecules return to the nucleus where the small-HDAg isoform supports further genome replication, and where both forms of hepatitis D antigen associate with new transcripts of genomic RNA to form new RNPs
- RNPs are exported to the cytoplasm where large-HDAg facilitates association with HBV envelope proteins in the ER to form new virus particles;
- these particles bud through an intermediate compartment;
- they are then exported from the hepatocyte via the trans-Golgi network to re-infect further cells.
Transmission of HCV
• Transmission
o Blood – transfusion, products, IVDU
o Sexual
o Sporadic
• Characteristics
o Only infects humans and chimps
o Icosahedral capsid, enveloped
o RNA genome (single stranded, +ve sense)
o 6 genotypes (genotype 1 most common in developed worlds hence where most action has been developed against)
Hepatitis C – Epidemiology
- 150-200 million chronic infections
- UK – approx. 214000 (Public Health England – HCV in UK 2015)
- 10-20% lead to cirrhosis
- Primary liver cancer implicated
Clinical consequences of HCV
• Course of infection:
o Onset 5-12w after infection
o Acute: often asymptomatic, raised ALT
o Chronic in 90% of cases
• Pathology
o Liver failure, cirrhosis, liver cancer
o 40% of Europeans with HCC have antibodies to HCV
o Damage is immunological (+ cytopathic)
HCV virion structure
• Capsid
o Icosahedral
o Single protein
• Envelope
o Two envelope proteins E1 and E2
o The virus forms a complex with serum lipoproteins to form VLDL + viral hybrids
These interactions cause difficulty in purifying the viral particle for investigation + the VLDL provides cloaking from immune system.
Which cell surface receptors does HCV bind to?
o CD81 o SR-B1 o CLDN1 o OCLN o Also the VLDL hybrid binds to the LDL-R on the surface and GAGs
Describe HCV infection of host cell
Receptor binding and interaction leads to invagination into the intracellular compartment – receptor mediated endocytosis
The coated vesicles are pre-lysosomal and have low pH
The change in pH confers conformational change in E (envelope) protein reveals a fusogenic domain
Often this domain is cleaved and released, which interacts with the vesicle to release the viral RNA into cytoplasm
Describe the HCV genome
- Single stranded RNA
- Approximately 9,600 nucleotides
- Positive polarity = large mRNA
- 5’ and 3’ UnTranslated Regions (UTRs) stem loop structure (important for protein-RNA interactions to occur)
o 5’-UTR Contain Internal ribosomal entry site (IRES)
Stem-Loop structure I-IV
IRES can be used to ensure that viral translation is active during periods when host translation is inhibited
o 3’-UTR
Has a Poly U tract (30-90 nucleotides)
Also incorporates stem loop structure
This 3’-UTR aspect is thought to bind to other parts of the genome and have a switch effect – causing it to either make proteins OR undergo replication (I.e. there is high affinity between HCV 3’ UTR and host ribosome, as well as interaction of 3’ UTR to 40S ribosomal subunit)
Describe the HCV ORF
• Single large Open Reading Frame (ORF)
o This translates into a big polyprotein, which is later cleaved to form the individual functioning proteins
o There are structural and non-structural proteins
Non-structural proteins are not packaged into the main virus, but are used to assemble it
RNA Replication in HCV
- Most of the polyprotein cleavage products (in particular NS3-5B) form the replicase complex associated with intracellular membranes
- Allows production of viral proteins and RNA in a distinct compartment
- The viral replication depends on making the complementary (-ve) copy, which is transcribed again to produce the original (+ve) strand
- The positive strand is 10x more concentrated than the negative due to its priority as the functioning strand
HCV Protein cleavage
- The large polyprotein contains all the viral proteins required
- The polyprotein is cleaved at junctions to produce the individual proteins
- Two classes of proteases are involved
o NS3 protease
Coded by the virus
Cleaves the non-structural domain
This is a potential antiviral target
o Host cell signalases
Cleave the structural part (mainly the envelope proteins)
These proteins have strong ER membrane association, where they bind to
These proteins can also bind to lipid droplets, which provides platform for the binding process
HCV ORF structure
see p60
HCV core protein
• Highly basic which makes up the viral nucleocapsid
o Binds viral RNA
o Associates with lipid droplets
- Nucleocapsid monomer binds to E1
- Associates at cytoplasmic side of ER
HCV envelope protein
• E1 (35 kd) and E2 (72 kd) implicated
• The envelope proteins are glycosylated – difficult to create immune response against them
• Two regions: homology region + divergence regions
• The divergence region is a hypervariable region (HVR) found on N-terminus of E2
o Lots of variants for this region
o Antibody development for one E2 will effectively wipe out viral population, but there are other mutated copies, which will repopulate hence why it is difficult to form a vaccine against HCV
• Functions o Receptor binding o Membrane fusion Can fuse the viral and host vesicular membranes together to release the viral capsin into cytoplasm during initial entry o Virion assembly
HCV NS3 (non-structural protein 3)
• Properties
o Protease (binds to NS4A as a cofactor)
Amino 1/3 of NS3
Serine protease with catalytic triad of amino acids
Cleaves HCV polyprotein both cis and trans
Targets host interferon stimulation pathway proteins viral persistence (by targeting specific host defences i.e. RIG-I and TLRs)
o Helicase in replication complex
Carboxy 2/3 of NS3
Unwinds double stranded RNA which is produced during viral replication
o ATPase (associated with the helicase activity)
• The viral polyprotein initially requires cleavage to produce NS3 from the non-structural domain
o How can it initially cleave itself without NS3 presence?
o Due to types of cleavage mechanisms: cis and trans
Cis-cleavage: intramolecular self-cleavage cleaves itself out of the polyprotein to begin the process (but this is less efficient)
Trans-cleavage: intermolecular cleavage occurs once mature protease has been cleaved out
• The helicase function is required to unwind the double stranded RNA during the viral replication
o However this occurs under a membranous web (proliferation of membranes from the Golgi apparatus) induced by NS4B this is where viral RNA and non-structural proteins complete RNA replication, so that the only particle released is positive sense single stranded RNA
This is an mechanism induced by the virus to hide from the host immune system (TLRs and RIG-I). Single stranded RNA is able to be detected, but to a lesser degree than double stranded RNA present in these webs. It also prevents nucleases from reaching and targeting RNA.
HCV NS5B
- RNA dependent RNA polymerase (RdRp) – replicates HCV viral RNA by using the positive RNA strand as template
- GDD motif (characteristic of RdRps)
HCV Replicase complex
• NS5B + NS3 + (NS4A) + (host proteins?)
• Membrane associated
o ER/perinuclear
• DNA stranded RNA localisation
Other non-structural proteins in HCV
• NS4A
o Protease complex with NS3 and its co-factor
o Localisation of RNA replication machinery to membranes
• S2
o NS2/NS3 protease
• NS5A
o Interferon inhibition – PKR
o P53 inhibition?
• NS4B
o Induces membrane web, which is a site of replication
o Inhibits cellular translation?
MicroRNA 122 (micR0122) and HCV
Lanford, Science, 2012
• Liver specific – hence why liver is a primary target for HCV infection
o Associated with miRISC this then binds to HCV 5’ UTR
• Required for HCV RNA replication as it stabilises and initiate process of replication
• It has been shown that siRNA against miR122 (small interfering RNA hence degrades miRNA) completely shuts off viral replication
o However, difficult to develop into an anti-viral therapy as it is not easy to deliver and has been shown to have numerous cellular functions (particularly regulating cholesterol metabolism)
Assembly and Release of HCV
• HCV produces double stranded RNA, which normally triggers a big IFN response via TLR pathway
• Therefore, the non-structural proteins form a membrane vesicle in the ER-lumen with a narrow entry point (membranous web)
o The narrow point allows diffusion of single nucleotides, but big immune related proteins cannot enter
o The assembly initiates on the cytosolic side of the ER membrane
• In the cytosol, the core protein is assembled which Is associated with lipid droplets (Lipid Droplet Model)
o The lipid droplet is brought in close proximity to the membranous web where replication is occurring .
o Core protein is released and interacts with RNA
o The lipid molecules are utilised to build the viral envelope + budding into the ER lumen
o This allows the encapsidation of the positive viral genome that is released into the cytosol for release
o The formed vesicles then fuse with the plasma membrane and release the virions from the cell (don’t forget: they will then associated with VLDLs to form viral hybrids)
Persistence of HCV Infection
- Site of RNA replication – ER vesicles
- NS3 – inhibits cell monitoring of virus molecules
- NS5A – inhibits interferon responses
- Variation in genome/protein structure – quasispecies
HCV evolution
• HCV RNA Polymerase – no editing mistakes (does not have function to stop it making mistakes)
o Error rate: 10-3 – 10-5 nucleotides/replication cycle (approx. 106 cellular mutation rate) – all occurring by chance (hence genes are not functional)
• BUT this allows the virus to mutate so that it is able to escape host immunity
• This means that host will create an antibody response to envelope proteins e.g. E2 and some virus will be neutralised but those with changes in amino acids in hypervariable region of glycoprotein E2 will escape
o This leads to new dominant genome to proliferate SEQUENCE DIVERGENCE
• Hence, a major problem for therapies e.g. HCV vaccine
Treatment for HCV
Vaccine
• E1/E2, NS proteins, subunit vaccines, DNA vaccines
• HCV is a quasispecies hence a major problem
• No vaccine so far
Antivirals
• Alpha interferon (pegylated allowed for increase in half-life of interferon)
• Ribavirin
o Daily treatment over 6-12 months
New Antiviral Protease Inhibitors for HCV
• NS3/4 protein – protease inhibitors
o Telaprevir
o Boceprevir
o Simeprevir
• Blockade of the NS3/4 protease would prevent assembly of replication machinery
o Secondary effect: blocking protease that downregulates the immune response hence boosting immune system as a secondary effect
• NS5A protein – polymerase inhibitors
o Sofosbuvir
• NS5 protein inhibitors
o Daclatasvir
But it is possible to get re-infected with hepatitis C especially if continuing high risk behaviour. Hence why a vaccine would be useful.
Consequences of Chronic HBV and HCV Infection
HCC
• HBV and HCV chronic infections (>30-50 years) predispose to HCC
• Potential Mechanism:
o Continuous liver damage
o Continuous repair and turnover of cells
o Accumulation of DNA damage
o Multi-hit transformation of cells
o HCC
Epidemiology of HBV and HCV
Hepatitis C – 2013
• 170 million infected worldwide
o Outdated: new WHO statistics show this number has been vastly overestimated and actually only ~70 million infection worldwide
• Diagnosis frequency estimated at 50%
• Estimates of 500,000 new infections per year
• Cumulative mortality rate of 5-7%
• Prevalence varies from 0.5-2% in Europe to 20% in Egypt
• Infection predominantly through iv drug use and non-screened blood supplies
Hepatitis B – 2005
• 400 million infected worldwide
• Evidence for 2 billion people exposed to HBV
• Estimates of 10-30 million new infections per year
• HBV is approximately 100 times more infectious than HIV
• Infection predominantly during childhood via blood contact
o One of the most infectious viruses that infect humans
Infection to Persistence in HBV and HCV
Both viruses can ultimately lead to liver failure due to cirrhosis and HCC. But the proportion of those who end up with these clinical manifestations is different for both viruses.
In HCV infection, 80% develop chronic disease –> 2/3s of these will develop progressive forms of the disease. (30% of which will develop into cirrhosis, 25% of which will result in liver failure, cancer, transplant or death)
In HBV, the proportion of those who develop an acute infection is heavily dependent on the age of the individual (90% of children = chronic, 5% of adults = chronic).
