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AdenovirusesHost defenses viral countermeasuresHost defenses, viral countermeasures,
gene therapy
Elizabeth Moran, Ph.D.Professor of Orthopaedics
My contact info: MoranEL@umdnj.edu Office: Cancer Center Room G-1200 Phone: 2-5854
November, 2008Viruses, Cells and Diseases
1
Office: Cancer Center Room G-1200 Phone: 2-5854
Adenoviruses: ChiefAdenoviruses: Chieffeatures
1. DNA genome.g
36,000 base pairs of DNA: small enough to be engineered effectively in the lab, and large enough to tolerate
~
incorporation of an expression sequence for a large mammalian protein without exceeding the capacity of its capsid structure.
CMV > Adeno > HPVCMV > Adeno > HPV10X 10X
2. Productive infection in a wide range of tissue types. 2
Clinical History ofClinical History of Adenovirus
Isolated from outbreaks ofIsolated from outbreaks of respiratory infections among military recruits in the 1950s
Over 50 serotypes are now known, divided into seven groups: A – G.
3Common lab serotypes (Ad2, Ad5) are type B
Ad i diAdenovirus diseases
The disease spectrum can vary depending on serotype; most common are cold symptoms, similar to rhinovirus infection.
Most people are sero-positive for adenovirus antibodies indicating they’ve had an infection. Adenoviruses are
id d i i l ll
Adeno can also cause eye infections.
widespread in animals as well.
In general, the most serious disease associated with Adeno is infant diarrhea, which can be severe, even life-threatening;
4
, , g;these cases usually involve the enteric serotypes Ad40 and Ad41.
Adenovirus diseases
Adenovirus rarely causes serious illness in healthy immuno-competent adults.
However, . . .
5
Ad14 is currently emerging as a virulent serotype capable of i f t l i i th i h lth lcausing fatal pneumonia in otherwise healthy young people.
6
Why is adenovirus so importantWhy is adenovirus so important to molecular biologists ?
It was soon identified as a tumor virus
It grows very easily in cell culture, so has become a model lab organismhas become a model lab organism.
7
Virus Interactions with the Host Cell
Adeno was instrumental in elucidating important concepts in the areas of:Adeno was instrumental in elucidating important concepts in the areas of:
i. Transcriptional regulation ii. Gene splicing as a mechanism to increase variability of gene
expression from a single gene.iii. Multi-step pathways to carcinogenesis and the interplay of
oncogene products and tumor suppressors.iv Host defenses and viral countermeasuresiv. Host defenses and viral countermeasures
iv Host defenses and viral countermeasures8
iv. Host defenses and viral countermeasures
Adenovirus structureAdenovirus structure
9Ad 5 binds to its receptor CAR (coxsackievirus and adenovirus receptor) through its fiber knob.
Adenovirus structureAdenovirus structure
Cell surface
Penton b
Fiber
Adenovirusvector
base
CAR integrin
Genetic engineering of the fiber protein
10
g g pis being used to improve viral delivery in gene therapy.
The adenovirus genomeDNA
The adenovirus genome is a linear length of aboutThe adenovirus genome is a linear length of about 36,000 base pairs
How much coding capacity is this ??
H thi h di it b thHow can this much coding capacity be the foundation for an autonomous life form ??
11What design principles can be used ??
Virus life cycle
1 Vi t th ll d t it DNA1. Virus enters the cell and uncoats its DNA.
2. Virus early genes are expressed. At least one gene must be able to get itself transcribed in theprevailing conditions of the cell. - -
- - so virus immediate-early promoters are frequently used
3. Viral DNA is replicated.
y p q yfor constitutive overexpression of exogenous genes inmammalian cells.
DNA replication requires the expression of large numbers of enzymes.
4. Capsid proteins are made and assembled around the DNA.
If the cell is not replicating, the virus has to activate the genes that encode these enzymes.
Immediate early gene Early genes Late genes
DNA Replication
12
y g
Timeline: 6-10 hours 8-24 hours 24-72 hours
Each capsid requires over 1600 protein molecules.
Virus life cycle
Role of the Early genes:y gi.Control expression of key viral and cellular genes
ii Counteract the ability of the cell to mount an anti viral responseii.Counteract the ability of the cell to mount an anti-viral response long enough to form new infectious units.
13Approximately 100,000 new viruses from a productively infected human cell
The adenovirus genome: E1A
Ad
E1A
Adeno genome
The E1A gene encodes two major products15
The E1A gene encodes two major products
E1A: seizes control of transcriptionE1A: seizes control of transcription
Two splice forms – same reading frame
13S TBP
Two splice forms same reading frame.
CR1 CR2 CR3CR4
13S
The 13S-unique region binds the TATA-Box Binding protein and is a promiscuous transcriptional activator – its main target is the other viral transcription units
12S
transcriptional activator – its main target is the other viral transcription units.
CR1CR4
12SCR2
16
Key host cell regulators oftranscription
pRBE2F
transcriptionfactors
CyclinsCDKs
DNA synthesis factors
300p300 c-myc
Tissue-specificgene
expression
17
Some immune response genes, e.g. intferon, MHC genes
E1A: seizes control of transcriptionE1A: seizes control of transcription
Two splice forms – same reading frame
13S TBP
Two splice forms same reading frame.
CR1 CR2 CR3CR4
3S
12S p300CR1
CR4
12S pRB CR2p300
18
The 12S form mainly targets cell cycle specific genes. Quiescent cells use the pRB protein to repress the promoters of cell cycle genes. E1A binds pRB to compromise its repression function.
But unscheduled DNA synthesis is sensed by the cell and triggers an
apoptotic response. . .
p53 activates apoptosis-related gene expression
The Bcl2 protein down-regulates the apoptotic response.
19
p53 induces transcription of the p21 cell-cycle i hibit d f t ti h Binhibitor, and of pro-apoptotic genes such as Bax.
Cyclin
p53 p21Cyclin
dependent kinases(CDKs)
Pro-apoptoticgenes
20
Bax-related proteins form a complex that creates pores in mitochondria releasing caspase-activating factorsmitochondria, releasing caspase activating factors.
Wh B i b d bDNA damage signal When Bax is bound by Bcl2, Bax cannot form a
functional complexp53
DNA damage signal
BaxBcl2
BaxBax BaxBax
Bcl2Bax
21
APOPTOSIS
The adenovirus genome: E1B
Ad
E1B
Adeno genome
The E1B gene encodes two major products22
The E1B gene encodes two major products
E1B 55K and 19K proteins prevent the cell f ti t t ifrom resorting to apoptosis
E1B: two splice forms – two different reading frames.
55K
19K
23
E1B 55KE1B 55K
p53Promoter of cell cycle inhibitor yor pro-apoptotic gene
p53
The 55K protein binds p53 and prevents it from activating cell cycle inhibitors or pro apoptotic gene expression
24
pro-apoptotic gene expression.
E1B 19KE1B 19K19K produced in abundance, sequesters most BAX
B l2 bi d dBcl2
19K
19K 19K
19K
Bcl2 binds and inhibits Bax
19K mimics Bcl22
B l2B
Bax
Bcl2Bax
Bax 19KBaxBaxBax
Bcl2Bax
Bax19KBax
BaxBaxBax
25
Now the DNA replication process is activated, and the cells cannot opt out by apoptosis - but p y p phow to get DNA replication to act on the viral genome and not the cell genome?
26
Th d i E2The adenovirus genome: E2
AdAdeno genome
E2AE2B
The E2 gene encodes three major proteins27
The E2 gene encodes three major proteins
E2 proteins: direct DNA synthesisto the viral genometo the viral genome
E2 encodes three proteins:80K pre-terminal binding protein (pTP)
attaches to 5” ends of viral DNA
72K DNA binding protein (DBP)binds to single stranded DNA – peels open the DNA for replication
140K DNA l140K DNA polymerase
Adeno DNA replication is primed by pTP,
28
Cellular DNA replication is discontinuous;requires RNA primers.
and uses its own DNA polymerase.
DBP coats the DNA to separate the strands.
E2 proteins: direct DNA synthesisto the viral genome
These three proteins help adeno capture all the work of the host cell’s DNA synthesis machinery.
Adenovirus can produce about 100,000 new copies of its DNA in an infected human cell
29
copies of its DNA in an infected human cell
Now you’ve replicated the viral DNA and are making hundreds of transcripts from the copies g p p- - -but the cell has mechanisms to restrict stability and translation of excess RNA
The interferon response evolved to recognize double-p gstranded RNA,
which is often associated with viral infection
30
Cellular measures to restrict viral RNA (i t f )RNA (interferon)
Interferon-induced RNA-dependent protein kinase (PKR) binds double-Interferon induced RNA dependent protein kinase (PKR) binds doublestranded RNA.
The kinase becomes activated by auto-phosphorylation ( ) when it binds as P
a dimer.
The activated kinase targets a translational elongation factor (eIF-2) for inactivation by phosphorylation, and thereby shuts down protein synthesis
PP
IF 2
Viral RNA
+PKR
PP= eIF-2
31
ProteinSynthesis
The adenovirus genome:The adenovirus genome:
The viral-associated (VA) RNAs
Ad
VA RNAs
Adeno genome
32
VA RNAs: competitively inhibit cellmechanisms to restrict RNA
Viral-associated RNA I and II are transcribed in great abundance.
And are too small to permit PKR to bind as a dimer, so PKR becomes sequestered in an inactive form. ~ 165 nucleotides
P
PKR becomes sequestered in an inactive form.
PP
33
Viral transcript
VA RNAs
VA RNAs: competitively inhibit cellmechanisms to restrict RNA
Viral-associated RNA I and II are transcribed in great abundance.
And are too small to permit PKR to bind as a dimer, so PKR becomes sequestered in an inactive form.PKR becomes sequestered in an inactive form.
34
Viral transcript
VA RNAsMathews and Shenk (1991) J. Virol. 65:5657-5662
mRNA degradation by small interfering RNAs
dsRNA miRNA
RNA interference involves a dsRNA cutting enzyme called Dicer, which cuts dsRNA made by viruses or formed from regulatory RNA Dicery g ysequences called microRNAs (miRNAs). Dicer cuts them into small pieces.
Dicer
The small dsRNA sequences unwind into small single strand RNAs (siRNA), which then combine with specific proteins to form an RNA induced silencing complex (RISC) Risc
small ds RNA
silencing complex (RISC). Risc captures complementary native mRNA molecules and cuts them up into untranslatable pieces.
RISCRISC
siRNA
mRNA
mRNA degradation by small interfering RNAs
dsRNA miRNA
Artificial siRNA sequences can be used to silence almost any gene.
DicerThis has tremendous potential clinical importance for gene therapy.
Dicer
small dsRNA
Can the the VA RNAs overwhelm the DICER or RISC enzymes
and impede siRNA therapy ?
RISC
and impede siRNA therapy ?
RISC
siRNA
mRNA
Aside from responses from the individual host cell the organism can also mount a systemiccell, the organism can also mount a systemicimmune response.
38
Cells sample newly manufactured proteins, cut them into p y ppeptides and present the peptides on the cell surface using proteins of the Major Histocompatibility Complex
Killer T cells can recognizeKiller T cells can recognize foreign peptides, can become activated to attack any cells presenting those
Infectedcell
y p gantigens. Viral
peptideantigen
MHC
An environment of lysing cells
T cell
An environment of lysing cells with associated release of interferons, inflammatory factors and cytokines, such as TNFα will
39
y ,activate killer T cells.
Th d i E3The adenovirus genome: E3
E3
Ad
E3
Adeno genome
The E3 gene encodes at least five small protein products40
The E3 gene encodes at least five small protein products
The viral antigens and MHC proteins are joined together in the endoplasmic reticulum on the pathway to presentation on theendoplasmic reticulum on the pathway to presentation on the cell surface
Viral peptide antigen
MHC
antigen
Endoplasmic reticulum
Golgi complex
Viral peptides
Proteosome
Viral protein
41
The E3 19K glycoprotein tethers the MHC proteins in the endoplasmic reticulum
E3 19KE3 19K
Endoplasmic reticulum
Golgi complex
Viral peptides
Proteosome
Viral protein
42So no viral antigens are presented on the surface
But even non-antigen-presenting cells can be subject to cytolysis mediated by cytokinessubject to cytolysis mediated by cytokines
such as TNFα emitted by natural killer cells
TNFR1
Caspase activation is triggered by the interaction of the TNF family of death ligands
FADD
Pro-Caspase 8
(TNFα, Fas ligand, TRAIL)….with their corresponding death receptors, such as TNFR1.
CASPASE 3
APOPTOSIS
43
The receptor cytosolic domains recruit adapter proteins, such as FADD, which then bind andactivate pro-caspase-8, which activates caspase 3 and causes cell death.
E3 proteins 10.4K and 14.5K associate to form a t i t li ti d d d ti (RID) lreceptor internalization and degradation (RID) complex.
Th RID lThe RID complex removes death receptors from the cell surface and marks them for degradation.
RID10.4K
14.5K
g
FADD
44E3 also encodes a 14.7K protein that protects further against TNF-induced cytolysis by inhibiting release of cytokines.
E3 DEATH Protein
Adeno resists apoptosis – but ultimately encodes an 11 6K nuclearAdeno resists apoptosis – but ultimately encodes an 11.6K nuclear membrane glycoprotein, regulated so as to accumulate only very late in infection, that has severe cytotoxic effect on cells;
. . . called the DEATH protein.
45Approximately 100,000 new viruses from a productively infected human cell
E4 proteinsE4 proteins
E4 expresses at least six open reading frames (ORFs) that are mostly not essential for viral growth in cell culture, though the gene is
i d f ffi i t i l th i i llrequired for efficient viral growth in primary cells.
E4_ORF6 associates with E1B_55K to block transport of cellular RNA out of the nucleus in favor of viral RNA.
Other E4 ORFs help in functions such as capturing cellular transcription factors for viral promoters, and targeting anti-viral host proteins for degradation
47
proteins for degradation.
Adenovirus early protein functions
E1A Takes control of viral and cell-cycle-related transcription.
E1B A 55 kDa protein binds and subverts p53A 19 kDa protein mimics Bcl-2
E d t l t f t i h ith l i di h t
E2 Encodes viral-specific DNA replication proteins, including a DNA polymerase
E3 Encodes at least four proteins, each with a role in evading host immune responses
Also encodes the DEATH protein, which accumulates to high l l l t i i f ti th i d ll l i t l tlevels late in infection, then induces cell lysis to release mature virus.
E4 Encodes about six small proteins with various functions that fine-tune interactions with host cellfunctions that fine tune interactions with host cell proteins.
VA RNAs Competitively inhibit interferon-induced shut-down of protein synthesis in response to double stranded RNA
In spite of all the virus countermeasures the host immunecountermeasures, the host immune
system usually does win out in the end
Most of us carry serum antibodies to adenovirus as evidence of past infections.
49
How does this affect the ability to use adeno for gene therapy?
Adenovirus gene therapyAdenovirus gene therapy
Can selectively replicating lytic viruses be used as anti-tumor agents ?
i.e as oncolytic agents
50
Hypothetical phenotype of an E1B (-) AdenovirusAdenovirus
In p53 (+) cells:In p53 (-) cells: p ( )
p53p53
Apoptosis would precedeViral assembly would
p53
p p pviral assembly.Infection would abort.
Viral assembly would Successfully precede lysis.Infection would continue.
NEIGHBORING CELLS would LIVENEIGHBORING CELLS would DIE
Adenovirus as a cancer therapeutic: Onyx -015 (H101)
Blue stain indicatesviral replication in the tumor;not in surrounding tissue,
i.e.: tumor cells (p53-negtive) die; normal cells (p53-positive) live.
Interestingly, though, the p53 state of the tumors does not really affect the outcome - - the specificity determinant may actually be the virus receptor - or the ability of tumor cells to complement- - the specificity determinant may actually be the virus receptor - or the ability of tumor cells to complement an E1B 55kDa-dependent function in progression from viral transcripts to translation.
A nearly identical virus, H101, was approved for human cancer treatment in China in 2006
Another oncolytic Adenovirus is based on an E1A mutation that makes E1A unable to bind pRB.
Adenovirus with E1A
mutation that makes E1A unable to bind pRB.
Adenovirus with E1A …unable to bind pRB
Virus cannot grow in these Vigrow in these cells
Virus can grow in, and lyse these cells
Normal cell pRB represses cell replication function Tumor cell
pRB is non-functional
Adeno-mediated gene therapy
FiberNon-lytic viruses can be used to deliver any desired coding
Penton base
Fiber deliver any desired coding sequence that can fit into the genome, if the target tissue has the proper receptors
CAR integrin
the proper receptors.
Potential uses:Potential uses:encode an enzyme to correct an inborn error
of metabolism or developmentencode an shRNA to target an unwanted
ll l
54
cellular gene.others ….
Adenovirus and gene therapy
An E1a-deleted virus will not express other viral proteins well –well
so a virus with this background can be used as a vector t d li h f i i h llto deliver a human gene for expression in a human cell without much effect from the virus –
this requires a propagation cell line that complements E1A: 293 cells
55What other coding units are dispensable ?
Where to fit in a new gene for adeno to express ?.
56
May lab stocks of adenovirus lack E3 –how does this affect use as a gene therapy vector?
Adeno carrying the HSV thymidine kinase gene can be used to activate the cancergene can be used to activate the cancer
chemotherapy pro-drug gancyclovir
CMVpromoter HSV thymidine kinase gene Adenovirus
vector
Viral kinase Cellular kinases
Gancyclovir
Toxic nucleotide
analogGancyclovirpro-drug
57DNA replication and cell proliferation blocked
This adenovirus is in phase 3 trials under the drug name Cerepro
How do you deliver your new gene therapy virus ?
Penton base
Fiber InhaleEat Inject
CAR integrin
Injectintra-tumorintravenous
integrin
Tumor cells tend not to have so many CAR receptors on their surface
58
Adeno faces very different challenges when injected IV rather than inhaled or eatenIV, rather than inhaled or eaten
In cell IntravenousIn cellculture
Intravenousinjection
In 1999, an 18-year old patient, Jesse Gelsinger, participated in a trial at UPenn’s Inst. of Gene Therapy aimed at treating inborn ornithine transcarbamylase deficiency. He received a high dose of an adenovirus vector, delivered systemically, and died of massive
f il tl ltorgan failure, apparently as a result of a severe immune reaction.
This tragedy delayed gene therapy trials for years, while greater consideration was given to safer
Heparinaseconsideration was given to safer viral delivery.
59HSPG = heparin sulfate proteoglycans LRP = lipoprotein related protein
Read for DiscussionRead for Discussion
Baker AH, McVey JH, Waddington SN, Di Paolo NC, Shhayakhmetov DM. (2007). The influence of blood on in vivo adenovirus bio-distribution and transduction. Mol. Ther 15:1410-1416
Bauzon M, Hermiston TW (2008). Exploiting diversity: genetic approaches to creating highly potent and efficacious oncolytic viruses. Curr Opin Mol Ther. 10:350-355.
Some interesting news articles:
Branca MA (2005) Gene therapy: cursed or inching towards credibility? Nature Biotech. 23: 519-521Nature Biotech. 23: 519 521
Garber K (2006) China approves world’s first oncolytic virus therapy for cancer treatment.J. Natl. Cancer Inst. 98: 298-300.
Osborne R (2008) Ark floats gene therapy’s boat for now
60
Osborne R (2008) Ark floats gene therapy’s boat for now.Nature Biotech. 26:1057-1059
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