Welcome
to the lecture series
Principles of
Virology
Thomas Kietzmann
Principles in Virology
Thomas Kietzmann
TK
Key terms and questions
What is DNA priming and how does it function?
Adenoviruses possess a linear genome; what is the mechanism
by which they overcome the shortening of the 5´ends during
replication?
Virus replication usualy requires a transcriptionally
active/growing cell. How does a herpes virus get around the
fact that a host cellis not transcriptionally active?
How is herpesvirus replication initiated and how is the
relatively large genome generated and packed?
In which phase of replication are viral capsid proteins
generated?
What is a latent HSV infection and what is a major
characteristic point for this?
What is acyclovir and how does it act?
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Principles in Virology
TK
Thomas Kietzmann
Viral DNA replication
Parvovirus
Hepatitis B virus
Retrovirus
VII
Adenovirus
Herpes simplex virus
Polyoma-, and Papilloma
Poliovirus
Reovirus
Rotavirus
Influenzavirus
Ebolavirus
Principles in Virology
TK
Thomas Kietzmann
Some history
• 1908 Discovery of chicken leukemia virus, Ellerman & Bang
• 1911 Discovery of Rous sarcoma virus, Peyton Rous (Nobel Prize 55 years
later)
• Called tumor viruses
Wilhelm Ellerman
Oluf Bang
Peyton Rous
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Principles in Virology
TK
Thomas Kietzmann
Tumor viruses
• Mainly found to have RNA genomes
• How could these viruses transform cells?
• 1970 Discovery of retroviral reverse transcriptase,
Temin & Baltimore; Nobel Prize 1975
Principles in Virology
TK
Thomas Kietzmann
Reverse transcriptase
• Enzyme that countered
Central Dogma: DNA => RNA => protein
• Retroviruses got their name because of their ability to reverse the
flow of genetic information
Retrovirus
Hepatitis B virus
VII
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Principles in Virology
TK
Thomas Kietzmann
Major insights
• Once reverse transcribed retroviral DNA is integrated into
host genome
• Becomes permanent part of host DNA
• Provirus
Principles in Virology
TK
Thomas Kietzmann
Simple retrovirus genome organization
U3
R
R
U5
U5
R
PPT
PPT
U3
R
• Monopartite, linear, dimeric, ssRNA(+) genome of about 7-11 kb, with a 5’cap and a 3’poly-A tail; coated with nucleocapsid protein
• There are two long terminal repeats (LTRs) at the 5’ and 3’ ends
• The LTRs contain R, U5, and U3 regions
• There are also a primer binding site (PBS) at the 5’end and a polypurine tract
(PPT) at the 3’end
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Principles in Virology
Thomas Kietzmann
TK
Retrovirus RNA dimer
• Explains why retroviruses are relatively resistant to UV and ionizing radiation
• Two copies of all genes
• Copy-choice rebuilds a functional genome
Principles in Virology
Thomas Kietzmann
Retrovirus life cycle
TK
•
Virus attaches to host receptors; TM
glycoprotein mediates fusion with cell
membrane
•
Internalization and partial uncoating
•
ssRNA(+) genome is copied into a linear dsDNA
molecule by the reverse transcriptase
•
Nuclear entry of the viral dsDNA which is
covalently and randomly integrated into the
cell’s genome by the viral integrase (=provirus
integration)
•
Transcription of provirus by Pol II produces viral
spliced and unspliced RNAs
•
Nuclear export of the incompletely spliced RNAs
•
Translation of unspliced viral RNAs produces
Env, Gag and Gag-Pol polyproteins
•
Assembly of the virion at the host cell
membrane and packaging of the viral RNA
genome
•
Budding, release of virions and proteolytic
processing of polyproteins by viral protease and
maturation of the virions
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Principles in Virology
Thomas Kietzmann
TK
Reverse transcriptase
• Primer can be DNA or RNA
• Primer must have paired 3’-OH terminus
• Template can be RNA or DNA
• Only dNTPs, not rNTPs, are incorporated
• Correct incoming dNTP is selected by base pairing
Principles in Virology
Thomas Kietzmann
TK
Reverse transcriptase
• Bacteria and Archaea have RT activity
• Therefore RT evolved before the separation of
Archaea, prokaryotes and eukaryotes
• RT might be the bridge between early RNA world
and modern DNA world
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Principles in Virology
Thomas Kietzmann
TK
Reverse transcriptase-RNase H
• Cleaves RNA only when in duplex form
• RNA can be in RNA:RNA or RNA:DNA duplexes
• Makes endonucleolytic cleavages
• Produces short oligonucleotides with 5’-phosphate, 3’-OH
Principles in Virology
Thomas Kietzmann
TK
Reverse transcription: priming
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Principles in Virology
TK
Thomas Kietzmann
Reverse transcription
-DNA +DNA
5’
+RNA -DNA
5’
3’
3’
3’ 5’
U3
R
R
U5
5’
5’
3’ 5’
DNA
synthesis
RNA
Hydrolysis
Jump
1
U3
DNA
Synthesis
3’
3’
DNA
synthesis
PB
tRNA
3’
RNA Jump
2
Hydrolysis
RNA 5’
Hydrolysis
PB
3’
Integration into
host genome
DNA
synthesis
5’
3’
3’
U3
R
U5
R
3’
3’
3’
3’
LTR
U5
3’
3’
5’
5’
LTR
5’ 3’
LTR = long terminal repeat; PB = primer binding; R = redundant; U = unique
Principles in Virology
Thomas Kietzmann
Retrovirus: Reverse transcription
TK
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Principles in Virology
Principles in Virology
Thomas Kietzmann
TK
Thomas Kietzmann
TK
Retrovirus: Integration of provirus
Retrovirus: Integrase
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Principles in Virology
TK
Thomas Kietzmann
Retrovirus: Integrase
3´-Processing
5´-ACTG
3´-TGAC
CAGT-3´
GTCA-5´
5´-ACTG
3´-AC
-GT
-GT
CA-3´
GTCA-5´
genomic DNA
´
CA-3 ´
-5
3´-A
C
5´
5´3´-
-3´
-5´
Strand Transfer
5 bp
Strand break 5bp gaps
5´3´-
3´-A
C
5´-ACTG
-AC
-3´
genomic DNA
5´3´-
-3´
-5´
CA
Preference for integration into DNA
sequences that are bent or underwound by
being wrapped around a nucleosome
genomic DNA
´
CA-3 ´
-5
3´-A
C
5´
5´genomic DNA
3´-
-3´
-5´
CA
GTCA-5´
-CA
genomic DNA
5´3´-
-3´
-5´
-CA
TG
-AC
CA
GT
-3´
-5´
Repair
genomic DNA
Principles in Virology
5´3´-
TG
-AC
Thomas Kietzmann
CA
GT
Integration of provirus
-3´
-5´
TK
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Principles in Virology
Thomas Kietzmann
Retrovirus gene transcription
TK
• A strong DNA promoter (the LTR) is built during reverse transcription
• The integrated DNA copy then directs the host transcription machinery to
synthesize many copies of viral mRNA
• The viral mRNA is translated into viral proteins OR encapsidated into
infectious virus particles
• No mechanism for precise excision of integrated provirus
• The only way to move the viral genome out of the cell is transcription by host
RNA pol II
• Genomes are liaered with ancient and modern retroelements
Principles in Virology
Thomas Kietzmann
Retroelements
TK
• Retrovirus-related sequences in cellular genomes
• When integrated into the germline = endogenous proviruses, ERV
• Often replication‐defective
• Nearly 50% of human genome comprises mobile genetic elements, including
endogenous proviruses and other retroelements
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Principles in Virology
Thomas Kietzmann
Retroelements
TK
Non-LTR containing retroelements
• LINES (long interspersed elements) represent the most abundant; They have
an unusual RNA polymerase II-promoter structure in which the promoter is
included within the final transcript. These elements create a polyadenylated
mRNA which codes for a bicistronic mRNA
• SINES (short interspersed elements) are independently derived from RNA
polymerase III-transcribed RNA genes (tRNAs and 7SL RNA; they are
transcribed by RNA polymerase III and encode a poly A, or A-rich region, at the
3′ end of the element
• Processed pseudogenes are derived from the mature mRNAs (spliced) from
numerous genes
Principles in Virology
Thomas Kietzmann
TK
Retrovirus gene transcription
Retrovirus genomes commonly ORFs
for proteins that can be found in the
mature virus:
• gag (group-specific antigen) codes
for core and structural viral
proteins
• pol (polymerase) (pol) codes for
reverse transcriptase/RNaseH,
protease and integrase
• env(envelope) codes for the
retroviral coat proteins
• Some viruses contain oncogenes
Multiple splicing events and posttranslational processing contribute to
increase the variety of proteins
expressed
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Principles in Virology
TK
Thomas Kietzmann
Discovery of oncogenes
1911 Virus as cancer causing agent
(Peyton Rous, 1966 Nobel price)
Chicken with sarcoma
v-src gene
(Harold E. Varmus,
1989 Nobel price)
Removal of tumor
Bacterial and cell-free filtrate
Injection of bacterial cell-free
extrat into healthy chicken
Chicken develops sarcoma
Principles in Virology
Thomas Kietzmann
TK
Discovery and Definition of Oncogenes
Oncos = gr. word for swelling
Peyton Rous,
(1966 Nobel price)
J. Michael Bishop, Harold E. Varmus
(1989 Nobel prize)
• The sarcoma causing part of the “Rous Sarcoma Virus“ was shown to
be the “sarc“ = v-src gene
• Uninfected, non-tumorigenic cells contain cellular homologues from vsrc and other retroviral genes
• Introduction of the term “proto-oncogene“ for these genes since they
appeared to be altered in tumors
Oncogenes are genes whose presence can cause uncontrolled
cellular proliferation and cancer
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Principles in Virology
TK
Thomas Kietzmann
Proto-oncogenes
• In all cells, control cell growth
• Highly regulated
• Normal cellular genes abbreviated as c-onc, encoded proteins are e.g.
c-SRC, c-MYC, C-RAS
• Certain retroviruses isolated from tumors carry encode altered copies
of c-onc genes abbreviated as v‐onc, e.g. v-src, v-myc, v-ras
Principles in Virology
TK
Thomas Kietzmann
Mechanism for oncogene capture
pol
Viral DNA
gag
env
Host DNA
Integration of provirus within a proto-oncogene
LTR
gag
pol
env
LTR
onc
Deletion
Wild-type mRNA
LTR
gag
pol
Deletion of virus and cell
sequences
Packing of deleted and wildtype genomes
Virus particle
LTR
gag
pol
v-onc
LTR
Non-homologoues recombination
during reverse transcription in newly
infected cell
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Principles in Virology
Thomas Kietzmann
TK
Defective vs non-defective retroviruses
• Defective viruses require helper virus to produce more virus
• Usually missing envelope proteins
• Envelope genes deleted during oncogene capture
Principles in Virology
Thomas Kietzmann
TK
Proviral DNA sequencs
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Principles in Virology
TK
Thomas Kietzmann
Six classes of (proto-) oncogene products are distinguished
1. Growth factors
(1)
(EGF)
(2)
-
(HER, NEU)
~
Receptors
~
2. Growth factor
(4)
(3)
(4)
Plasma
membrane
3. GTPases in
signal transduction
(5)
(RAS)
Nucleus
4. Tyrosine Protein Kinases
(SRC, ABL)
(5)
5. Ser - Thr - Protein Kinases
(RAF, ERK)
(6)
6. Transcription factors
4
(FOS, JUN, MYC)
Principles in Virology
Thomas Kietzmann
TK
How can a viral infection transform a cell?
• Cytopathic effects must be reduced or eliminated
• -The infected cell does not die
• -Viral replication must be reduced or eliminated
• -Transformed cells do not produce virions
• The cell must continue to divide
• -It becomes immortal
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Principles in Virology
Thomas Kietzmann
TK
What happens to the viral genome in transformed cells?
• Some transformed cells contain all or parts of viral genomes integrated
into the host genome
• Sometimes no viral nucleic acid remains in the transformed cell
These were key, but mystifying early observations
Principles in Virology
Thomas Kietzmann
TK
Retroviruses transform cells by three mechanisms
• Rapid tumor formation: e.g. RSV; 2 weeks
- RSV has activated dominant oncogene in genome (v-SRC)
- Protein produced immediately when virus replicates
• Intermediate kinetics of tumor formation: e.g. ALV; months
- ALV carries no dominant v-onc gene
- cis-activation: provirus turns on expression of endogenous
oncogene
• Slow kinetics of tumor formation; e.g. HTLV; years
- HTLV carries no dominant v-onc gene
- Does not cause cis-activation of local oncogenes
- A viral regulatory protein activates oncogenes by trans-activation
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Principles in Virology
TK
Thomas Kietzmann
Retroviruses transform cells by three mechanisms
Principles in Virology
TK
Thomas Kietzmann
Rous Sarcoma Virus (RSV)
1911
Cell-free extract from a chicken sarcoma
induces a tumor after injection into healthy chicken
(Peyton Rous, Rockefeller Institute, New York; 1966 Nobel price)
Principle: Virus
RNA
Integrase
(pol)
Membrane protein
(env)
Capsid proteins
(gag)
cap
Reverse transcriptase/RNaseH
(pol)
Virus
R U5 PB 
gag
pol
env
src
U3 R
(A)n ss (+) RNA
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Principles in Virology
TK
Thomas Kietzmann
Rous Sarcoma Virus (RSV)
cap
Virus
R U5 PB 
gag
pol
src
env
U3 R
gag
(A)n
pol
cap
(A)n
env
src
cap
v-src mRNA
Myr
QLLPACVLEVAE
1
1
ds DNA
integrated
U3 R U5
U3 R U5
cap
(A)n ss (+) RNA
SH2
SH3
SH1
c-src mRNA
cap
Intron
src
KINASE
533
FLEDYFTSTEPQYQPGENL
Y
src
Exon
(A)n
cellular
ds DNA
c-src proto-oncogen
Principles in Virology
(A)n
526
TK
Thomas Kietzmann
Function of the c-src and v-src protein
Deletion
e
PM
i
Phospho tyrosine
SH3
P
Protein tyrosine kinase
(SH1)
Regulatable
activity
c-src
Constitutive
activity
v-src
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Principles in Virology
Thomas Kietzmann
TK
Growth Factor Signalling Cascade (Part 1)
1. Growth Factor
Binding
Dimerisation

Helix
EGF; PDGF
Autophosphorylation
ATP ADP
Tyrosine
Kinase
2. Growth Factor Receptor
Phospho tyrosine
P
P
P
P
P
P
SH-Domäne
2
GTP
GTP
SH-Domäne
3
Protein
P Protein
GDP
GRB2 GEF Ras
ADAPTOR (SOS)
TyrK
Src
Signal
transduction
3. Signal Transduction Proteins
EGF = Epidermal Growth Factor; PDGF = Platelet-Derived Growth Factor;
GRB = growth factor receptor binding protein; GEF = guanine nucleotide exchange factor (= sos, son of sevenless);
Ras = rhabdo myo sarcoma; PLC= Phospholipase C, SH= src homology; src = cytosolic tyrosin kinase
Principles in Virology
Thomas Kietzmann
TK
Hepatitis B Virus: variations on the retrovirus theme
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Principles in Virology
TK
Thomas Kietzmann
Incidence of Hepatitis B Virus Infections
Principles in Virology
TK
Thomas Kietzmann
HBV particle
Envelope proteins
Membrane protein (Host)
PreS1-HBsAg gp42
PreS2-HBsAg gp36
HBsAg gp27
cap
Capsid protein
HBcAg p22
P protein p90
Membrane lipid bilayer (Host)
partial ds DNA
Terminal protein/
Reverse transcriptase/
RNase H
The genome consists of two DNA strands, a longer negative-sense strand and a shorter and positive-sense strand of
variable length; Replication involves an RNA intermediate
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Principles in Virology
TK
Thomas Kietzmann
Partial ds DNA virus: Hepatitis B-virus (HBV) gene expression
Env proteins
PreS1-HBsAg
Secretory protein
PreS2-HBsAg
HBeAg
HBsAg
EcoR1
(-)
1
(+)
3’
3,2 Kb
cap
different
frame
Capsid protein
(A)n
(A)n
(A)n
3’
5’
Nucleoproteins
P protein
cap
Reverse transcriptase
RNase H
Terminal protein
HBcAg
HBc,e,sAg = Hepatitis B core, early, surface Antigen
Principles in Virology
Thomas Kietzmann
Hepatitis B-virus (HBV) replication
•
•
•
•
•
•
TK
Virus attaches to host receptors through
major surface antigen
Relaxed circular DNA (RC-DNA) and
capsid are transported via microtubules
to the nucleus where DNA is released,
and repaired to form covalently closed
circular DNA (cccDNA)
Transcription by RNA polymerase II of the
pregenomic
RNA
(pgRNA)
and
subgenomic mRNAs, inducing synthesis
of all the viral proteins
pgRNA is encapsidated, together with
the P protein, and reverse-transcribed
inside the nucleocapsid in (-)DNA
covalently linked to P protein
(+)DNA synthesis from the (-)DNA
template generates new RC-DNA.
Transport to the nucleus of new RC-DNA
leads
to
cccDNA
amplification;
alternatively, the RC-DNA containing
nucleocapsids are enveloped and bud as
virions
No genome integration, only upon rare non-specific recombination events
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Principles in Virology
Thomas Kietzmann
TK
Reverse transcription of the HBV pregenomic RNA
pgRNA is covalently linked to P protein
Four nucleotides are synthesized by P protein near the stem
loop close to the 5′ end of the pgRNA
The nascent minus-strand DNA exchanges template to an
acceptor sequence that overlaps the 3′ copy of the 12-nt
direct repeat DR1.
Minus-strand DNA synthesis resumes after template exchange, resulting in a genome-length minus-strand
(light blue). Degradation of pgRNA by RNase H activity of P protein generates the 18-nt RNA primer for plusstrand DNA synthesis
Principles in Virology
Thomas Kietzmann
TK
Reverse transcription of the HBV pregenomic RNA
• The 3′ terminus of the primer is complementary to
DR1
• Primer translocation. The plus-strand primer
exchanges template from DR1 to DR2, which is
near the 5′ end of the minus-strand DNA
• Plus-strand DNA (darker blue line) is initiated
from DR2 and elongated until the 5′end of the
template
• The minus-strand template is terminally
redundant for 7 or 8 nt (5′r and 3′r)
• Circularization. The nascent plus-strand moves
from 5′r to base pair with the 3′r. This switch
circularizes the genome
• Elongation of the plus-strand after circularization
results in a RC DNA genome.
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Principles in Virology
TK
Thomas Kietzmann
Hepatitis B-virus: Protein components
mRNA
Protein
kb
MG
AA
Function
10 + 183 - 32
= 161
183
3,35
HBeAg
p16
3,3
HBcAg
P protein
p22
p90
2,4
PreS1-HBsAg
p39
gp42
128 + 55 + 226
= 409
Envelope protein
Induction of neutralizing Ab
Adsorption to receptor
2,1
PreS2-HBsAg
p33
gp36
p24
gp27
55 + 226
= 281
226
= 226
Envelope protein
Induction of neutralizing Ab
Envelope protein
Induction of neutralizing Ab
HBsAg
0,8
HBx-Protein
Secretory protein
Capsid protein
Terminal protein/
Reverse transcriptase/
RNase H
p17
Principles in Virology
Transactivator
TK
Thomas Kietzmann
Serum Diagnostics: Acute Hepatitis B
Hepatitis*
Infection
Transaminases
HBV-DNA
Anti HBc-IgG
Serumlevel
HBsAg
Anti-HBc-IgM
Anti HBs-IgG
HBeAg
Anti HBe-IgG
0
1
2
3
4
5
6
7
8
9
10
11
12
Month
*Symptome: Jaundice, Hepatomegalie
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Principles in Virology
Thomas Kietzmann
TK
Key terms and questions
What What are the three kinds of biochemical reactions carried
out by the enzyme reverse transcriptase?
What molecule serves as primer for the first strand cDNA
synthesis by reverse transcriptase?
What is the function of the retroviaral geome sequence named
PBS?
What kind of functions may be encoded by v-onc genes?
How is the proviral DNA of a retrovirus different from the RNA
genome?
What is frequently absent in a replication defective retrovirus?
Hepatitis B virus is a DNA virus with partially gapped genome;
why does this virus require reverse transcriptase?
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