This lecture is about Virology of HCV presented by Dr. Mahmoud Elzalabany, Internal Medicine Resident, Ahmed Maher Teaching Hospital. The lecture was presented in the scientific meeting of Internal and Tropical Medicine departments, Ahmed Maher Teaching Hospital titled (Towards Eradication of HCV in Egypt) in celebration of World Hepatitis Day on July 28, 2016. https://www.facebook.com/AMTH.IM https://www.facebook.com/events/1072758396145209/ http://www.no4c.com

1. Virology of HCV
By
Mahmoud Elzalabany
Internal Medicine Resident, AMTH

2. Discovery of HCV
• Until 1975, only two hepatitis viruses had been identified, Hepatitis A virus
and Hepatitis B virus.
• However, HAV and HBV were excluded from being the cause of
approximately 65% of post-transfusion hepatitis.
• These hepatitis cases were termed “non-A, non-B hepatitis” (NANBH).
• Inoculation of chimpanzees with blood products derived from humans
with NANB hepatitis led to persistent increases of serum ALT indicating
that an infectious agent was the cause of the disease (Alter 1978).
• NANBH agent could be inactivated by chloroform (Feinstone 1983) and
was able to pass through 80 nm membrane filters (Bradley 1985).

3. Discovery of HCV
• These findings suggested that the NANBH causing agent would be a small
virus with a lipid envelope.
• Lack of a suitable cell culture system for cultivation of the NANBH agent
and limited availability of chimpanzees prevented further characterization
of the causative agent for several years.
• In 1989, using a newly developed cloning strategy for nucleic acids derived
from plasma of NANBH infected chimpanzees, the genome of the
causative agent for NANBH was characterized (Choo 1989)
• The virus causing NANBH was subsequently termed hepatitis C virus
(HCV).

4. Hepatitis C Virology
• HCV is an enveloped RNA virus
belonging to the genus hepacivirus of
the Flaviviridae family.
• HCV has a spherical shape, 50 nm in
diameter.
• It has smooth outer surface and spike
projections formed of E1 and E2
proteins.
• The outer layer surrounds the spherical
nucleocapsid consisting of the HCV core
(C) protein
• Inside the capsid is the viral genome.

5. Viral Genome
• Hepatitis C virus genome consists of single-stranded positive-sense RNA
molecule of 9600 bases and serves as a messenger RNA (mRNA) for the
translation of viral proteins.
• The RNA molecule contains a single open reading frame (ORF) of about
9000 nucleotides coding for a precursor polyprotein of about 3000 amino
acids.
• The ORF is flanked by 5’ and 3’ nontranslated regions (NTR) at each end.

6. Viral Genome

7. Viral Genome
• The 5’NTR is formed of about 340 nucleotides and contains the internal
ribosome entry site (IRES) that initiates the cap-independent translation of
HCV genome.
• The 3’NTR is principally involved in negative-strand priming during HCV
replication.
• The nucleotide sequence variability is distributed throughout the entire
viral genome. The 5’UTR is the most conserved region in the genome
while the regions encoding envelope proteins (E1, E2) are the most
variable ones.

8. Viral Proteins
• The precursor polyprotein resulting from HCV RNA translation is cleaved
by viral and host enzymes into three structural proteins (core, E1, E2) and
seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B).
• The structural proteins are essential components of the HCV virions,
whereas the non-structural proteins are involved in RNA replication and
virion assembly.

9. Viral Proteins
Viral Proteins Functions
Core It forms the viral capsid that contains the HCV genome and has regulatory functions.
E1 and E2 These envelope glycoproteins are responsible for adsorption of the virus to receptors on the host cell plasma
membrane.
p7 It is a membrane protein which forms ion channels and plays an essential role in virus infection.
NS2 The NS2 and NS3 proteins form a cysteine protease which catalyzes the cleavage of the polyprotein precursor
between NS2 and NS3.
NS3 & NS4A The NS3 and NS4A proteins form a serine protease which is responsible for cleavage of the remaining HCV
polyprotein. The C-terminus of NS3 has NTPase/helicase activity required for viral replication.
NS4B The NS4B is an integral membrane protein. It appears to be responsible for the formation of the HCV RNA replication
complex.
NS5A The NS5A protein is a membrane-associated phosphoprotein that has multiple functions in HCV RNA replication, viral
assembly, and virion release.
NS5B NS5B serve as RNA-dependent RNA polymerase responsible for HCV replication. It lacks a proofreading mechanism
leading to the conservation of misincorporated nucleotides. Together with the high rate of viral replication, this leads
to continuous HCV evolution.

10. Direct Acting Antivirals
Group Drugs
HCV NS3/4A Protease Inhibitors •Boceprevir
•Telaprevir
•Simeprevir
•Paritaprevir
•Asunaprevir
•Grazoprevir
HCV NS5B polymerase inhibitors •Sofosbuvir (Nucleotide analogue)
•Dasabuvir (Non-Nucleoside analogue)
HCV NS5A replication complex
inhibitor
•Daclatasvir
•Ledipasvir
•Ombitasvir
•Elbasvir
•Velpatasvir

11. HCV Life Cycle

12. Adsorption and viral entry
• The HCV lifecycle begins with the attachment of a virion to specific receptors on
hepatocytes.
• Tetraspanin CD81, scavenger receptor B type I (SR-BI), tight junction protein
claudin-1 (CLDN1), and occluding (OCLN) are some known cellular receptors for
HCV attachment.
• This process may be mediated by VLDL or LDL that is reported to be associated
with HCV virions in human sera.
• After binding with its receptor, the virion is internalized by endocytosis.
• The acidic environment within the endosomes trigger fusion of the viral envelope
with the endosome membrane followed by release of the viral RNA into the
cytoplasm of the cell.

13. Translation and post-translational processing
• The HCV RNA binds to the 40S and 60S ribosomal subunits forming the
translation complex at the endoplasmic reticulum.
• Translation of HCV RNA ORF results in a 3000 amino acids polyprotein
precursor.
• The precursor polyprotein is processed by four proteases.
• The cellular signal peptidase (SP) cleaves the core protein, E1, E2, and p7.
• HCV encodes two viral enzymes responsible for cleavage of the remaining
non-structural proteins.
• The NS2/NS3 cysteine protease cleaves the junction between NS2 and
NS3, while the NS3/NS4A serine protease cleaves the remaining proteins.

14. HCV RNA replication
• HCV RNA-dependent RNA polymerase (NS5B) is the key enzyme for viral
RNA replication.
• NS4B forms an ER-derived membranous web containing most of the
nonstructural HCV proteins.
• NS3 has a helicase activity that facilitates HCV RNA replication.
• The NS5B uses the positive-strand HCV RNA as a template for the
synthesis of a negative-strand RNA. The later is used in turn to synthesis
numerous positive-strand RNA to be used as genomic RNA for HCV
progeny as well as for polyprotein translation.

15. Assembly and release
• After the viral proteins and the HCV RNA have been synthesized, these
components have to be arranged in order to produce infectious virions.
• The HCV assembly and release process is not fully understood.
• It appears to be closely linked to lipid metabolism.
• HCV infection induces a profound change in the intracellular distribution
of lipid droplets (LDs) from generalized cytoplasmic pattern in uninfected
cells to accumulation around the perinuclear region in HCV-infected cells.
• The virion is a lipoviroparticle with a lipid composition that resembles
VLDL and LDL with associated apoE and/or apoB, which are essential for
the infectious virus assembly.

16. HCV Genotypes
• There is a high genetic diversity in HCV genome with about 6 major genotypes that
differ at 30-35% of nucleotide sites and 67 confirmed subtypes differing at <15% of
nucleotide sites.
• HCV genotypes show a large variability in geographic distribution.
• Genotype 1 is the most prevalent (46.2%), followed by genotype 3 (30.1%), while
genotypes 2, 4, 5 and 6 represent the remaining 23.7% of HCV cases.
• Genotypes 1 and 3 dominate in most countries, while genotypes 4 and 5 dominate
in low income countries.
• In Egypt, 91% of HCV cases are of genotype 4.
• This variation represents a challenge in developing vaccines and pan-genotypic
treatments for HCV.

17. HCV Genotypes

18. Thank You!