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M229: Cell Biology and Pathogenesis
Molecular and cellular biology of herpesvirus pathogenesis
February 2002
Ren Sun
[email protected]
1. Introduction of herpesviruses
2. Mechanism of herpesviral replication
3. Herpesviral latency
4. Transformation by Epstein-Barr virus
Discussion of viral oncogene.
Take-home messages:
1. The purpose of viral life is to replicate itself. (its associated
pathogenesis or human diseases are secondary effects)
2. Virus utilizes host machinery to replicate.
(cellular and organal)
3. The ultimate form of parasitization is to co-exist with the host.
(at both individual and population levels)
4. Studies of viruses have double meanings.
(medically important and scientifically interesting)
5. Herpesvirus has two phases of the life cycle:
latent infection and lytic infection.
herpein: to creep (Greek)
Herpesvirus particle
Electron cryomicroscopy and 3D reconstruction of HSV-1 B-capsids
Z.Hong Zhou et al. Science, 2000 April
Virion structure of herpesvirus
1. Herpesvirus particle is composed of an icosahedral
capsid, containing a large linear double-stranded DNA
genome.
2. Viral genome is a double-stranded DNA (150 kb to
250 kb), encoding about 100 genes. Only 40-60% of the
genes are essential for viral replication in tissue culture.
3. The capsid is surrounded by a tegument and wrapped
in a lipid envelope. The tegument, which consists of viral
proteins, is unique to herpesviruses.
4. The particle is about 200 to 300 nm with 100 nm capsid.
Herpesviruses
• Large genome, complex gene expression and
regulation
• Ubiquitous infections, primary infections
usually in-apparent in childhood
• latent/persistent/recurrent infections
• Transition between lytic and latent replication
• Associated with acute and chronic/malignant
diseases
Human herpesviruses
Name Common name associated diseases
subfamily
HHV-1 Herpes Simplex Virus 1 (HVS-1)
a
Oral, ocular lesions; encephalitis
HHV-2 Herpes Simplex Virus 2 (HSV-2)
a
Genital oral lesions; neonatal infections
HHV-3 Varicella Zoster virus (VZV)
a
Chickenpox, shingles
HHV-4 Epstein-Barr virus (EBV)
g
Infectious Mononucleosis; tumors (BL, NPC, NHL)
HHV-5 Human Cytomegalovirus (HCMV)
b
Congenital infection; systemic infection
HHV-6 Human herpesvirus 6
b
Exanthem subitum; systemic infection
HHV-7 Human herpesvirus 7
b
Exanthem subitum?
HHV-8 Kaposi’s Sarcoma associated herpesvirus
g
Kaposi’s Sarcoma, B cell lymphomas
size
150kb
150kb
130kb
170kb
230kb
160kb
160kb
140kb
Herpesvirus family
highly co-evolved with host
widely disseminated froom human to flog, fish and snake
• Alphaherpesviruses
– Fast replication, cytolytic; latent in neurons (Herpes
Simplex, Varicellar Zoster)
• Betaherpesviruses
– Intermediate replication, cytomegalic; latent in
glands, kidneys (Cytomegalovirus, HHV-6, -7)
• Gammaherpesviruses
– Slow replication, latent in lymphocytes,
lymphoproliferative, (Epstein-Barr virus, HHV-8)
What is Latency? and why there is latency?
At cellular level, Latency is the reversibly nonproductive
infection of a cell by a replication-competent virus.
(1) They can successfully evade the host immune response.
(2) They enable their genome to persist in the latently
infected cell, thereby in the host.
(3) They co-exist with the host cells and the host.
(4) The host becomes a active carrier for transmission.
All herpesviruses are capable of establishing latent infection.
Please note: The difference with abortive infection or infection with
defective virus.
Mechanisms to establish latency
1. HSV infects non-dividing cells such as neurons.
No protein expression.
2. EBV infects dividing or mitotic cells such as B cells.
A set of viral protein(s) and origin of DNA replication initiation is
required for replicating viral genome.
Function of partition is required to retain the viral genome in
daughter cells during the latent infection.
3. Use RNAs to avoid antigenicity.
4. Down-regulation of presentation of viral antigen(s).
Life cycle of herpes simplex virus
“Fields Virology”
HSV-1 infection and
lytic replication
HSV-1 latency and
reactivation
“Fields Virology”
A Model of EBV Life Cycles in vivo
Primary infection
of epithelium
Secondary infection
of epithelium
Lytic
Reactivation
10 infection
of B cell
III
III
III
Resting B cell
III
III
III III
I/II
Latency switch?
Latency switch?
III III
Kill
Resting T cell
III
Kill
Memory CTL
10 CTL response
Primary infection
20 CTL response
Persistent infection
Viral entry of cell
(simplex virus)
(1) Virus attachment to the host cell.
envelope glycoproteins (gC and gB) binds heparin sulfate
(2) Binding of viral surface protein (gD) to one of the cellular
receptors (herpesvirus entry mediators, Hve)
HveA (the tumor necrosis factor receptor family)
HveB and C (nectins, Immunoglobulin superfamily)
(3) Fusion of the virus envelope with cellular membrane.
gI and gE appear immediately on surface after entry
Multiple interactions during binding, penetration and uncoating.
(2) Virus attachment to the host cell.
EBV envelope glycoprotein gp350/220 binds to CD21(type 2
complement receptor, CR2) directly, in a fashion similar to that of
C3d of complement.
Activation of tyrosine kinase signal transduction, activate the B cell.
gp42 (BZLF2) binds to HLA class II (MHC class II) as co-receptor.
gp85/gp25/gp42 oomplex mediates membrane fusion.
HHV-7 binds to the CD4.
CD4 is not by itself sufficient for entry.
HHV-7 also can infect certain CD4-negative cells.
Each herpesvirus uses different viral envelope protein to bind
different cellular receptor.
Receptors for other herpesviruses are to be identified.
3) Penetration and uncoating of the virus particle.
For HSV-1, this occurs via a process of fusion of the viral
envelope with the cell plasma membrane. HSV-1
glycoproteins gB, gD, gH and gL involve in the fusion.
Capsid binds to microtubules and is propelled by dynein
(microtubule-dependant motor). Capsid moves to nuclear
pore. Binding to nuclear pore complex causes conformation
change of the capsid and injection of genome into nucleus.
Functions of some virion proteins in the tegument:
Host shut-off protein (vhs)
Transcriptional activator (VP16)
Protein kinases
Viral mRNAs in virions of HCMV (Bresnahan et al Science 2000)
Herpesvirus genomes organization
Isomers
e.g.Channel catfish herpesvirus
1
e.g.Human herpesvirus-8
e.g.Epstein Barr virus
e.g.Pseudorabies virus
1
1
2
e.g.Herpes simplex virus
4
Circularization and replication of viral genome
For herpes simplex virus, the linear genome ends are held
together by viral protein and immediately ligated by cellular
enzymes (via recombination process). The genome remains
nonnucleosomal during lytic replication (nucleosomal during
latency).
For EBV, active DNA synthesis is required over 24 to 36 hours.
The DNA is replicated by viral DNA polymerase during lytic
replication in a mechanism similar to rolling-circle model. Initiated
at specific location(s) by viral protein(s).
Gammaherpesvirus (EBV and HHV-8) DNA is replicated by
cellular DNA polymerase during latent infection in a mechanism
similar to plasmid replication in E.coli. A viral protein Initiates the
replication at a specific location and partition to daughter cells.
Viral DNA replication in the lytic infection
A. Replication origin.
1. Origin of DNA replication during the lytic cycles,
initially learned from defective particles.
2. A/T rich palindromic sequences,
located near transcription initiation sites.
3. At least two lytic origin in each wild type virus.
One is sufficient in vitro.
B. Mechanism of replication
1. Theta form at the early phase.
2. DNA recombination converted to rolling-circle model
with extensive branches.
3. The final products are head-to-tail concatamers.
Cleavage and package of viral genome
1. Lytic replication and terminal repeats are essential.
2. pac 1 and pac 2 sequences, located at the each end of the genome,
are conserved in most herpesviruses.
3. Each herpesvirus has a different terminal sequence arrangement.
Rolling circle replication
Lytic replication
Infection &
circularization
Pac 1
Pac 2 Pac 1
Pac 2
Egress
Cleavage
& package
Isomerization of HSV genome:
High efficiency and specificity, equal mole after one round of
infection. Defect does not affect in vitro viral replication, but all wild
isolates can isomerize.
a b
UL
b’ a c US
c’ a
1:1
Replication and Isomerization
1:4
1:4
1:4
1:4
Lytic DNA replication and cleavage of gamma-herpesvirus genome
Vector
I
12
Flag-Rta
Rta
Rolling-Circle Replication
24 36 48 12 24 36 48 12 24 36 48 hr p.t.
Multimer
HindIII
Monomer
I : infected BHK cells
Probe
Maturation of virion:
A. Capsid:
1. Capsid is assembled in the nucleus. Empty capsid can selfassemble in vivo and in vitro (from purified proteins).
2. DNA is packed into pre-assembled capsids containing scaffolding
proteins, proteinase and other viral proteins.
3. Head-full mechanism to measure the length of the DNA.
4. Terminal repeat sequences (pac1 and pac 2) are required. Processing
massive and inter-connected, non-linear DNA. Capsid proteins
binding to viral DNA, probably no histones. 70,000 spermidine and
40,000 spermine per capsid.
B. Envelope:
1. Envelop at nuclear membrane, de-envelop in cytoplasm and re-envelop
at cell membrane.
2. There are few cellular proteins and a lot viral proteins in envelope..
C. Tegument:
1. 20 to 40 viral proteins (some are well organized & attached to capsid).
2. Some RNAs, eg HCMV.
Cellular changes during lytic (productive) infection
1. Deposition of materials (tegument proteins) on nuclear
membranes or into nucleus. Insertion of viral glycoproteins
into cytoplasmic and plasma membranes.
2. Nucleolus enlarged, displaced toward the nuclear membrane
and dis-aggregated later.
3. Fusion of infected cells with un-infected cells to form
polykarycytes (polykarycytosis). Syncytium is common to
many viruses.
4. Destabilization of all mRNAs by a tegument protein vhs (virion
host shot-off),which removes preexisting host mRNAs. Vhs
induce endoribonucleolytic cleavage. Host protein synthesis
are shot-off at initial infection to ensure the expression of
viral immediate-early genes.
Cellular changes during lytic (productive) infection
5. Viral immediate-early gene products stimulate
cellular metabolism (eg. Inactivate p53 & Rb).
6. Host DNA and protein synthesis shot-off during viral
DNA replication, to ensure the synthesis of viral
DNA and structure genes.
7. Apoptosis is actively inhibited by viral functions in all
stages of viral replication (encoding Bcl2, inactivating
Rb & p53), but the cell is ultimately destructed.
8. A late protein g34.5 is required in neuron cells to prevent
triggering of cellular stress responses that result in a
premature total shut off of protein synthesis.
Gene expression Kinetics of HSV
Hours post-infection
0
2
4
5
7
10
Peak agene Peak bgene
expression expression
Immediate-early
15
Late (ggene
expression
Late
Early
DNA replication
Virion assembly
20
DNA viruses inactivate p53
After viral infection, p53 becomes activated and induces the
apoptosis pathway. Most DNA viruses have evolved mechanisms to
inhibit p53.
SV40 Large T-antigen binds to the p53 DNA-binding domain,
preventing p53 from binding to its control elements in DNA.
Ad E1B 55K stimulates p53 DNA-binding, but it also contains a
strong repression domain, so that it turns p53 from an activator into
a repressor of genes regulated by p53.
HPV E6 protein binds to p53 and induce its degradation via
ubiqutination and proteosome degradation.
EBV Zebra inactivates p53 by direct binding. EBV Rta binds to Rb.
HHV-8 LANA inhibits the trans-activation function of p53.
(Friborg et al, Nature, 1999)
Cellular targets of DNA tumor viruses
Ad, SV40, HPV, EBV, HHV-8
E1A, T,
E7, Rta, Rta?
M
G2
G1
Rb
E2F
G1 Cdk-Cyclins
S
Cki1
Ad
SV 40
HPV
EBV
HHV-8
E1B
T
E6
Zebra
LANA
p53
EBV induces
HHV-8 encodes
Bcl2
Apoptosis
Ad
EBV
HHV-8
E1B
bcl2
bcl2
Expression and Functions of Herpesviral Genes
Latent genes Suppressers of immune recognition,
Activators of transcription & proliferation
+
?
agenes
Immediate-early
+
_
bgenes
Early
+
Transcription activators,
Suppressers of immune recognition
_
ggenes
Late
Non-structural regulatory proteins,
Enzymes for viral DNA replication,
Inhibitors of apoptosis
Major structural proteins,
Regulatory factors in virion (VP16, vhs)
DNA replication enzymes
• Defining characteristic of herpesviruses (reactivation
from resting cells).
• Early (b) genes encode 2 groups proteins which
increase DNA synthesis
– Increase DNA replication (viral DNA polymerase,
single-stranded DNA binding protein,
helicase/primase, etc)
– Increase nucleic acid metabolism (thymidine
kinase, ribonucleotide reductase, dUTPase)
T hy midine
Thymidine
Kinase
d TMP
dT DP
dT T P
DNA
Dihy dr of olat e
Thymidylat e
dUMP
met hylene
dUTPase
dUDP
dUT P
Ribonucle ot ide
Reduct ase
UDP
UMP
dCT P
Dihy drof olat e
Reduct ase
Synt hase
THF
T HF
Human herpesvirus-8 encoded cellular homologues
ssDNA binding protein
DNA Polymerase
DHFR
TS
TK
Alkaline Exonuclease
Helicase-primase
Uracil DNA glucosidase
dUTPase
Ribonucleotide Reductase, small
Ribonucleotide Reductase, large
Complement Binding Protein
IL-6
vMIP1 (macrophage Inflammatory Protein)
vMIP2
vMIP3
Bcl-2
Interferon Response Factor-1
Cyclin D
FLIP
OX-2
IL-8 receptor
AP-1 like transcriptional activator
myb like transcriptional activator
In simplex virus, nearly 50 % of viral genes are not required for replication
in fibroblast cells in culture.
Gene expression cascade during lytic replication
$
Virion
VP16
+
+
_
a-genes
+
_
b-genes
+
_
+
_
g-genes
The balance between latency and lytic replication
$
VP16, vhs
latent-genes
Latency
Virion
+
Lytic replication
+
_
a-genes
+
_
b-genes
+
_
+
_
g-genes
Latency of herpes simplex virus
1. Viral DNA exists as a circular double stranded DNA in the
nucleus of neuron cells for life time until reactivation.
2. LATs (latency-associated transcripts, discovered by Jack
Stevens at UCLA, are the only RNAs expressed during
latency. 2 kb LATs are circular RNAs, resulted from
splicing of a 8.3 kb primary transcript. Function is not
clear.
3. LATs are non-coding nuclear RNAs. No viral gene proteins
are expressed or required to establishing latency!!??
4. The molecular mechanism of HSV latency and reactivation
is not clear.
Epstein-Barr virus (gammaherpesvirus)
• Lytic infection in epithelial cells of
nasopharynx and salivary gland
• Latent in B lymphocytes
• Multiple copies of viral episome in nucleus
are replicated synchronously with cellular
chromosomal replication.
• Sets of latent gene products (proteins and
RNAs) contribute to viral episome replication
and cell proliferation.
Epstein-Barr virus and cancer
•
Nasopharyngeal carcinoma
– One of the most prevalent malignancies in East Asian
– Antibody responses to EBV antigens
– EBV genome always found in tumor cells
•
Gastric carcinoma (NCNT)
– 1000 % in NCNT 1
– 10 % in general gastric carcinoma
– EBV genome always found in tumor cells, high a-EBV IgA
•
Hodgkin’s lymphoma
– Widespread in W Europe and US
– EBV DNA in Reed-Sternberg cells (~50% cases)
•
Burkitt’s lymphoma
– Common in Africa, EBV always found in endemic BL tumor cells
– EBV genomes (parts) found in some sporadic BL
•
Immunoblastic B-cell lymphomas
– Common in the immunosuppressed patients
– EBV genome always found in tumor cells
Evidence for EBV is a tumor virus
(First human tumor virus, 1964)
• Sero-epidemiology, elevated antibody titers:
aVCA (IgA), aEA (IgG)
• Viral genome and expression in tumor cells
• Monoclonal viral genome in tumor cells
• Transform primary B cells in vitro
• Cause tumor in new world monkey
• Prevent and cure of immunoblastic
lymphoma by CTL specific for EBV antigens
Cleavage and package of viral genome
1. Lytic replication origin and terminal repeats are essential.
2. pac 1 and pac 2 sequences, located at the each end of the genome,
are conserved in most herpesviruses.
3. Each herpesvirus has a different terminal sequence arrangement.
Rolling circle replication
Lytic replication
Infection &
circularization
Pac 1
Pac 2 Pac 1
Pac 2
Egress
Cleavage
& package
Lytic DNA replication, cleavage package and circularization of
gamma-herpesvirus genome
Multimer
Transformation &
clonal tumor
Monomer
HindIII
Establishment of latency in primary B cells (in vitro):
Virology
1. Activation of B cells by binding of virions to the receptor CD21.
2. Circularization of genome requires (repair) DNA synthesis.
3. The copy number per cell increases in the first week to
about 10 to 50 per cell without lytic replication, and remains
stable thereafter. Viral genome is replicated, once and only once,
by cellular DNA polymerase in early S phase. The only viral
protein required for maintaining latent replication is EBNA1
(Epstein-Barr viral nuclear antigen 1).
4. The viral genome undergoes progressive methylation,
except the OriP region, which contains multiple binding sites for
EBNA1 and is the nuclear matrix attachment
site and transcription regulation sequences for latent RNAs.
Transformation of primary B cells (in vitro)
1. Six viral nuclear antigens (EBNAs) are expressed. Many
of them have transforming activities.
2. Three cytoplamic membrane protein (Latent membrane
protein, LMPs) have transforming activities.
3. Two novel RNAs, Epstein-Barr virus encoded RNAs
(EBER1, EBER2) are expressed to over one million copies
per cell. Good diagnosis marker for EBV related diseases.
Transforming activity?
4. The latency can be disrupted and switch to lytic replication.
The frequency is dependent on the host and can be
enhanced by TPA, sodium butyrate or a-IgM antibody
crosslinking. The switch is controlled by transcription factors
Zebra and Rta. The lytic genes are expressed in a cascade.
Establishment of latency and transformation of
primary B cells (in vitro, at least three steps)
Cell biology:
1. Activation of primary B cells by binding of the receptor.
2. Induce B cell gene expression and proliferation in the
way similar that in responses to antigens, mitogens or IL-4 or
a-CD40. The cells are dependent of cytokines and high cell
density. This is the results of the expression EBNA2 and
LMP1 (type II latency).
3. The expression of other EBNAs and LMP2s leads to
full transformation. EBV in these cells induces express of
bcl-2, activates the signal transduction pathways of TRAFs,
Notch, NFkB and inactivates Rb.
Latent genes of Epstein Barr virus
• 6 Epstein-Barr virus Nuclear Antigens (EBNA’s)
– EBNA 1 maintains viral episome via OriP.
– EBNA 2, 3A, 3B, 3C and LP involve in proliferation
via Notch signaling pathway. (Hsieh, Science 1995, PNAS 1999)
• 3 latent membrane proteins (LMP’s)
– LMP1 activates membrane signaling (TRAFs)
activate proliferation and block apoptosis.
– L MP2 A&B sequester B cell receptor-associated
tyrosine kinase (a-IgM reactivates EBV), inhibit
reactivation from latency. Not directly contribute to
transformation, but necessary.
• 2 small RNAs (EBERs): transformation?
Activation of TRAF signal transduction pathway by EBV LMP1
TNFR
TNFR
TNF
TRAFs
TRAFs
NFkB
Growth
LMP1
EBV infection
TRAFs
NFkB
Growth
Notch signal transduction pathway
Delta
Notch
Notch
Protease cleavage
Notch IC
Notch IC
HDAC
SAPCIR
Notch IC
X
CBF/Su
Without ligand binding, CIR recruit SAP
and HDAC which represses transcription.
CBF/Su
After ligand binding and cleavage, Notch IC with an
activation domain, enters nucleus, replaces CIR and
activates transcription.
Activation of Notch pathway by EBV
Notch
Notch
Notch IC
Notch IC
EBNA3
EBNA2
CBF/Su
CBF/Su
EBNA2 with an activation domain, enters nucleus,
replaces CIR, release the suppression and activates
transcription.
EBNA3A&C binds CBF, prevents DNA
binding and releases the suppression.
Gene expression regulation during latency
EBERs are always expressed.
Type I === use of Qp.
Expression of EBNA1 only, no CTL recognition. EBNA1 is essential forgenomic
Maintenance.
Type II === use of Qp and LMP2p
Expression of EBNA1 and low levels of LMP2s, weak CTL recognition.
Type III=== use of Cp, Wp and LMP1&2p.
Expression of all six EBNAs, and higher levels of LMPs. Presenting
strong transforming activities and strong CTL epitopes.
Circular genome
EBERs
Cp
Wp
LMP1p LMP2p
Qp
TR
EBNA1
LMP1
LMP2
Switch among different latent forms and lytic cycle
Latency I
EBNA1
EBERs
Latency II
Latency III
EBNA1
EBERs
EBNA1, 2,
3A,3B,3C,LP
EBERs
LMP1,2s
LMP1,2s
Lytic cycle
p53
Rb
Zebra
Rta
$
Virion
A Model of EBV Life Cycles in vivo
Primary infection
of epithelium
Secondary infection
of epithelium
Lytic
Reactivation
10 infection
of B cell
III
III
III
Resting B cell
III
III
III III
I/II
Latency switch?
Latency switch?
III III
Kill
Resting T cell
III
Kill
Memory CTL
10 CTL response
Primary infection
20 CTL response
Persistent infection
Similarity of Gammaherpesvirus Genome Organization
MHV-68
HHV-8
EBV
4-11
17-50
4-11
17-50
4-11
17-50
20
40
52-69
*
80
52-69
*
72-75
75
52-69
*
60
72-75
80
100
120
140
160
Kb
Genomic difference among
gammaherepesviruses
*
bcl-2
M8
tRNA 1-8
M5,K3,M6
M1-4
MHV-68
ORF72 cyclin D
ORF73 LANA
ORF74 IL-8R(GPCR)
ORF50 Rta
M10a,b,c
M7
4-11
17-50
52-69
*
M12-14
72-75
PAN
IL-6,DHFR,K3,TS,
MIP-II,K5,MIP-I
bcl-2
K1
HHV-8
4-11
LMP1,2
BARF1
BALF2
EBV
vIRF,K10-11
Zebra
17-50
BILF1
BILF2
4-11
EBNA1
17-50
20
40
OX-2
80
52-69
*
Zebra,BZLF2
EBNA3a-c
gp350/220
BLLF2
72-75
EBER1-2
BHRF1,EBNA2,EBNA4,vIL-10,LMP2
75
52-69
*
60
K12,FLIP
80
100
120
140
160
Kb
KSHV gene expression program
Latency
Latent
+
Lytic replication
Immediate-early
Cyclin D
+
FLIP
Rta
n-butyrate
LNA
TPA
Early
Late
PAN RNA
sVCA
vIL-6
vMIP-I
+
vMIP-II
vMIP-III
vIL-8 receptor
Bcl-2
DHFR
TS
TK
Mechanism controlling the balance between latency and lytic replication of KSHV
Latency
Lytic replication
NFkB
p65
+
Latent
genes
Rta
+
+
PKC
+
+
PAN RNA, vIL-6
&
Other lytic genes
The life cycle of gammaherpesvirus
$
Transformation
Tegument
Virion
cell death
Lytic cycle
Latency
Rta
a-genes
+
b-genes
g-genes
Biological implication of complicated gene
expression regulation of EBV
Multiple level of regulation:
The switch between type I, II, and III latencies;
The switch between latency and lytic replication.
Purposes:
Evade the immune system, maximize production of
virions or proliferation of infected cells and minimize damage
to the host.
Conclusion:
The viral gene expression regulation plays a critical
role in determining the biology and pathogenesis of EBV
Infection.
Take-home messages:
1. The purpose of viral life is to replicate itself. (its associated
pathogenesis or human diseases are secondary effects)
2. Virus utilizes host machinery to replicate.
(cellular and organal)
3. The ultimate form of parasitization is to co-exist with the host.
(at both individual and population levels)
4. Studies of viruses have double meanings.
(medically important and scientifically interesting)
5. Herpesvirus has two phases of the life cycle:
latent infection and lytic infection.
herpein: to creep (Greek)