(RT) dsDNA Viruses Flashcards
Baltimore classification of RT dsDNA viruses
dsDNA RT viruses belong to Class VII, according to the Baltimore classification of viruses
These are viruses that have the capacity to replicate their genome via reverse transcription
RT DNA viruses differs from group I (dsDNA) and class II (ssDNA) by their capacity to replicate via RNA intermediate
Examples of RT DNA viruses
hepatitic B virus (HBV)
cauliflower mosaic virus
see slides
Hepadnaviruses: Introduction
The hepadnaviruses are classified into the
family Hepadnaviridae. • The viruses in the group are so named
because they are DNA viruses which infect the
liver cells of the host causing hepatitis. Hepatitis – DNA – Viruses (HepaDNAviruses)
• Hepadnaviruses are commonly known as
hepatitis B viruses (HBVs)
How are Hepadnavirsues unique
These viruses are unique in two ways:
- They have a small genome which is efficiently
organized to encode virus proteins and regulate the
expression of virus genes. - Hepadnaviruses (RT DNA) replicate their genome via
RNA intermediate using reverse transcription
enzymes. This differentiates them from dsDNA viruses
which replicate their DNA directly to DNA.
Note: Hepadnaviruses and the plant virus (cauliflower
mosaic virus) both form a group referred to as the
Pararetroviruses
The hosts of Hepadnaviruses
Some hepadnaviruses infect mammals
including humans (e.g. hepatitis B virus, a
major cause of disease and death).
• Some infect birds (e.g. heron HBV and
woodchuck hepatitis B virus, duck HBV, non- pathogenic virus).
Human Hepatitis B Viruses (HBVs)
prevalence and distribution
Prevalence and distribution: The virus is
estimated to infect about 350 million persons
worldwide
• Most infected person are either from Asia,
while some from Africa.
• There is high prevalence of infection in the far
north of Northern America and Greenland
which is usually overlooked due to their small
population.
Hepatitis B Viruses (HBVs) continued
Transmission of HBVs
• The virus is mainly found in the blood and semen of
infected persons. Thus, similar to that of HIV.
• HBVs have resulted in about 50 million new infection
annually.
• Prenatal infection: Majority of infected children
acquire the virus from their mothers.
• Also, the use of contaminated syringes and needles for
injections account for a large number of infections in
developing countries.
Hepatitis B Viruses (HBVs) continued
• Symptoms:
Symptoms: Most HBV-1 infected persons show
little (mild) or no symptoms, particularly in
children.
• However, infection is more persistent in new born
than adults as 90-95% of infected children were
observed to harbour the virus for a long period
of time compared to 1-10% of adults.
• Some infected persons with persistent infection
may remain healthy, while some may develop
severe hepatitis, resulting to cirrhosis and liver
cancer.
• HBVs result in about 500,000 deaths annually.
HBV Virion
4 major components
The shape of the virion is fairly spherical,
having a diameter of approximately 42 nm.
• The HBV virion has 4 major components:
- DNA
- P (polymerase protein)
- Capsid
- Envelope
HBV DNA
HBV has a small genome (3.2 kbp), composed of
dsDNA, with one strand incomplete, making the DNA
partly single stranded or partly double stranded.
• Also, a short sequence is triple stranded, due to
complementary sequence at the 5’ ends which gives
the DNA a circular conformation.
HBV P (polymerase protein)
Plays important role in replication. Thus, every
HBV virus has one P protein at least.
• The N terminus of P protein forms a ’’terminal
protein‘’ domain, separated by a spacer from the
reverse transcriptase domain.
• The C terminus of P protein has a ribonuclease H
domain (RNase H).
• Additionally, the P-protein has a DNA-dependent
DNA polymerase activity.
HBV Capsid
HBVs possess icosahedral shaped capsid
containing pores, with short spikes protruding
from the virion surface.
Figure 3 : HBV capsid:
The capsid in composed of dimers of the C
(core) protein, which is mainly alpha- helical.
The C terminus of the C protein is simple
due to a large amount of arginine; hence,
the region function in binding virus
genome.
HBV Envelope proteins
The HBV envelope has three protein species named small (S), medium (M) and large (L).
• The M and L proteins are the longer versions of S
proteins.
• The S proteins is the most abundant among the
three proteins.
• Each of the three proteins has one or more
glycosylation site.
• The hepatitis B surface antigen (HBsAg) is
composed of the surface regions of the envelope
proteins.
N terminus of L protein
binding site for cell
receptors is situated close
to the N terminus of the L
protein.
half of the
L molecules have the N
terminus on the outside of
the envelope
the other 50% have the N terminus on the inside of the envelope, attached to the capsid.
HBV Non-infectious particles
HBV are unique by their ability not only to replicate
progeny virions but also huge amount of noninfectious molecules that have been released from
infected liver cells.
• These non-infectious particles do not contain
nucleocapsid, but lipids and virions envelop proteins.
• The shape of some of the particles are either sphere or
filament.
• Both the virions and non-infectious particles are more
abundant in the blood than in the liver.
• Although, the non-infectious particles (mostly spheres)
are produced in large number than virions.
Figure 5: HSV noninfectious and virion
particles
Note that the non- infectious particles are produced in large amount, especially spheres than virions. Both non-infectious particles (spheres and filament) have diameters of 22nm , while the the filaments have variable length which could reach 200nm
Importance of non-infectious particles
in HBV
• Immune evasion: A potential reason for the
HSV producing large number of non-infectious
particles could be to ‘’deceive’’ the host
immune cells against itself (virion) as the
immune cells are likely to be attracted to the
more abundant non-infectious particles than
the virus itself.
HBV soluble protein
• Hepatitis B e antigens (HBeAg): HBVs are also
able to release its protein (HBeAg) in the blood
along side the non-infectious particles.
• HBeAg is like the C protein, except that it possess
10 extra amino acid residues at the N terminus,
while the C terminus lacks 34 amino acid residues.
• The HBeAg is suspected to play a role in host
immune evasion similarly as the non-infectious
particles.
HBV Genome
• The HBV genome is one of the smallest among viruses with
a size of 3.2 kb.
• The genome has four ORFs, which result in the translation
of seven proteins.
• Implies that the genome encodes an enormous amount of
information for replication. Gene efficiency is achieved by:
- Each nucleotide in the genome is maximized for protein
coding and capable of reading more than 50% of its
genome in two reading frames. - Coding capacity is maximized by expressing the L protein
in two conformations with different roles. E.g, the outer
part of the L protein binds to host cell receptors while the
inner portion of it binds the capsid with the nuclear
envelope (see figure 4).
Figure 6: HBV Genome
he genome has 4 ORFs (P, C, S, X). The complete (-) strand and incomplete (+) strandare surrounded by these 4 ORFs
The P ORF makes up about 80% of the HBV
genome, occupying the entire region of S ORF, while overlapping the C and X ORFs.
DR1 and DR2 are sequences with direct
nucleotide repeats present in the genome.
L proteins: Are synthesized from translation of
complete pre-S1- pre-S2 – S ORFs.
fM proteins: synthesized from translation of pre-S2
– S ORFs. S protein: synthesized from translation of S ORFs
The HBV Genetic groups
and where are they most present
Figure 7: A phylogenetic tree showing the
HBV Genetic group
Molecular biology techniques have enabled
the sequencing of the genomes of HBVs
into 8 genetic groups (genotypes A – H)
Also, gene sequencing have revealed
relationship between the human HBV and
those in other primates. The various HBVs show fairly geographic
restrictions; genotypes A is prevalent in
Northern Europe while B and C
predominates in Asia.
Replication steps in HBV
and what sort of cell type do they infect
• HBV infect mainly the liver cells (hepatocytes) in
hosts. Generally, the steps in HBV replication are:
• Attachment
• Entry
• Transcription
• Translation and post-translational modifications
• Nucleocapsid Assembly
• Genome replication
• Envelope acquisition and exit of virion
Attachment of HBV to host cells
Mechanism of attachment of HBV to host cell
is still under investigation, however, some
have been proposed as follows:
• That annexin V and polymeric IgA are
suspected to play a role. • The HBV binds to host receptors through a site
on the L proteins.
Entry of HBV from cytoplasm into nucleus
• Entry of HBV is through endocytosis through shedding
of the envelope, fusing of the nucleocapsid with the
plasma membrane, then the endosome membrane.
• The fusion facilitates the release of the nucleocapsid in
the cytoplasm.
• Microtubules: Transport cell invading virions to the
nuclear pore where they gain entrance into the nucleus.
• Some scientific evidence suggest that the nucleocapsid
can pass through the nuclear pore. In this case, the
capsid is shed in the nucleus. However, its still
uncertain if the capsid is shed on the nuclear pore,
releasing only the genome into the nucleus for
transcription.
Figure 9: Entry and release of HBV into the
nucleus and conversion into cccDNA
On entry into the nucleus, the genome is
transformed into a circular DNA molecule.
The P protein is released from the 5’ end of the
(-) strand.
DNA synthesis at the 3’ end makes the entire
genome double stranded.
The ends of each strand are ligated to form a
covalently closed circular DNA (cccDNA). Note the following:
- It is still unclear whether the above DNA
modification is mediated by host cell
enzymes or virion proteins - Replication of virus DNA doesn’t occur in
the nucleus but some are released back
into the nucleus during replication.
Transcription in HBV
• The template for transcription is the cccDNA. • Transcription in liver cells: A minimum of 2 of the
4 promoters (pre-S1, pre-S2, X and pre-C regions)
are highly specific for liver cells and some of the
transcription factors involved liver proteins. • Transcription is performed by RNA polymerase II,
resulting in the synthesis of four types of RNA
with varying sizes. • All four transcripts are capped at 5’ end and
polyadenylated at the 3’ end.
Transcripts outcomes in HBV
he four size classes of RNA transcripts are shown in green (0.9 kb, 2.1 kb, 2.4 kb and 3.5 kb) Some of the larger RNAs belonging to the 3.5kb class (green and blue outer circles) will serves a mRNAs while other will function as pregenomes (replication of virion genome)
V
Figure 1
two subsets of 3.5 kb vary in size
among the members of the class:
The shorter subset acts as mRNA for
synthesis of C and P proteins and as
pregenomes.
The longer subsets is translated to
become the HBeAg which is released
from the cell.
Also, there exist two subsets of the 2.1 kb mRNAs: The longest subset is translated into M proteins, while the shorter subsets is translated into S proteins.
Assembly of x in HBV
Capsid:
Pregenome RNA:
Packaging signal:
Figure 12: Assembly in HBV Capsid: The C proteins form dimers and are assembled into capsid. Majority of HBV capsids are made up of 120 dimers while a few are built from 90 dimers or less
Pregenome RNA: formed from the
fusion of one molecule of P protein, together with several cell proteins and
a molecule of 3.5 kb RNA.
Packaging signal: The P protein
together with a short sequence at the
5’ end acts as signal for packaging
pregenome into the capsid.
HBV genome synthesis
• DNA synthesis in HBV occurs in the newly assembled capsid.
• Conversion of RNA to DNA: DNA is synthesized from
pregenome RNA via reverse transcriptase domain of P using
mucleotides that enter the capsid through it holes.
• Synthesis of (-) DNA: The terminal protein domain of P acts
as a primer for activating the synthesis of (-) DNA.
• At the initial stage, activation results in a 4-nucleotide
sequence of (-) DNA, after which DNA synthesis continues
at the 5’ end of the pregenome template.
• RNase activity of P proteins degrades the pregenome RNA
from the RNA-DNA duplex, resulting in the removal of all
RNA except for a remnant of 15-18 bases including the cap.
• Synthesis of (+) DNA: The –OH located at the 3’ end of the
RNA remnant acts as primer for the synthesis of (+) DNA.
2 fates of nucleocapsid in HBV
• In the course of (+) DNA synthesis, a nucleocapsid can
undergo 2 pathways.
- Migrate back to the nucleus to increase the pool of
cccDNA to function as template for transcription. - Undergo maturation events leading to budding off
through the membrane between the endoplasmic
reticulum and Golgi complex where it acquires virion
envelope proteins.
• Exit of the nucleocapsid through the membrane
terminates DNA synthesis as it is cut off from the
nucleotide pool in the cytoplasm, hence the reason
why the (+) DNA
Figure 13: Functions / destination of progeny HBV nucleocapsid:
In the course of (+) DNA synthesis, a nucleocapsid can either migrate back to the nucleus to increase the pool of
cccDNA to function as template for transcription or
Undergo maturation events leading to budding off through the membrane between the endoplasmic reticulum and
Golgi complex where it acquires virion envelope proteins. Source: Adapted from Carter and Saunders 2013; Virology: Principles and Applications, 2nd Edition
Figure 14: Exit of progeny HBV and
other non-infectious particles from
cells
During budding, the nucleocapsid binds to copies of the N termini of L protein. The virions are transported to the cell surface where they leave the plasma membrane through exocytosis along with non-infectious proteins (spheres and filaments)
Pathology of HBV in liver cells
• The infected liver cells of the host may not
immediately be destroyed; some may survive for
a number of years releasing large quantities
progeny virions and non-infectious particles.
• Most cases of damaged liver cells arouse not
from the direct effect of cell damage due to HBV
in the cells but from the death of HBV-infected
hepatocytes due to immune killing (attack)
Prevention and treatment of HBV
infection
vaccines
• Earlier vaccines was composed of non-infectious
spheres and filaments extracted from the blood
of HBV donors.
• Recent vaccines are formulated from the gene of
HBV S protein using recombinant yeast cells.
Prevention and treatment of HBV
infection
drugs (Lamivudine)
Drug (Lamivudine): Is a nucleoside analog, which
suppresses virus replication with little side effects.
It is administered orally and cheaper that other
vaccines.
