Transcript Proteomics of Poxvirus - KEIVAN BEHESHTI MAAL'S HOMEPAGE

IN THE NAME OF GOD
Department of Microbiology, Islamic Azad
University, Falavarjan Branch
Advanced Virology
dsDNA Viruses
By:
Keivan Beheshti Maal
1
POXVIRUSES
Large Enveloped dsDNA
Viruses
Genus Orthopoxvirus
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buffalopox virus {buffalo, cattle, human}
camelpox virus {camel} (CMLV)
cowpox virus {rodents, felines, bovines, human}
ectromelia virus {mousepox}
monkeypox virus {rodents, primates, human}
rabbitpox virus {colonized rabbit only}
raccoonpoxvirus {North America}
skunkpox virus {North American striped skunk}
taterapox virus {African gerbil}
vaccinia virus {no natural reservoir}
Uasin Gishu disease {Central African horses}
variola virus {human; eradicated from nature}
volepox virus {California pinon mouse and voles}
Poxvirus
 Variola (smallpox) virus is most closely related to
camelpox
 Both are believed to have evolved from a common
ancestor
 probably a rodent poxvirus
 5,000-10,000 years BC
Virus Structure
 most complex of the viruses that infect animals
 dry weight vaccinia contains
 90% protein
 100 proteins
 functional enzymes
 polymerases, kinases,
 ligases etc.,
 structural proteins
 5% lipid
 3.2% DNA
Virus Structure
 Among largest, most
complex animal viruses
 brick-shaped membrane
bound viruses
 Non-obvious helical or
icosahedral symmetry
 360 x 270 x 250 nm
 Size of chlamydia under
light microscope
 complex internal
structure
Virus Structure
 "core"
 biconcave = dumb bell shaped
 tightly compressed nucleoprotein
Virus Structure
 Core
 Linear double-stranded DNA genome
 terminal hairpin loop
advantage?
 several tandem (i.e. direct) repeat sequences
 ends of the genome form direct repeats
 inverted terminal repeats (ITRs).
Virus Structure
 Core DNA
 most essential genes located in the central part of the genome
(highly consereved)
 e.g assembly and replication
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non-essential genes are located at the ends (much more diverse)
e.g genes encode unique biological determinants:
Host range
Virulance factors
Evasion from immune system
 General size:
 130-300kbp
 Vaccinia
 190,000 nucleotide base pairs
 completely sequenced
Virus Structure
 CORE
 Enzymes
 Host RNA polymerase is in the cell nucleus
 Pox replicates in cytoplasm
(poxviruses use a virally-coded DNA -dependent RNA
polymerase needed immediately upon infection)
 in virions
 flanked by 2 "lateral bodies"
 function unknown
 surface of virus covered with
filamentous protein
Virus
Structure
 envelope
 intracellular particles only have an
inner membrane
 IMV - intracellular mature virions
 not host membrane
 extracellular forms contain 2
membranes
 EEV - extracellular enveloped virions
 second derived from Golgi or ER
Proteomics of Poxvirus
 Proteomics:
The protein composition of virion
Aims:
1) important prerequisite for functional studies
2) Important prerequisite for studying
pathogenicity responsible proteins
Proteomics of Poxvirus
 Poxvirus gene classes:
 Early genes:
1) early and 2) immediate early
(most of the core enzymes)
 Intermediate genes
 Late genes:
1) virion structural proteins
2) morphogenesis factors for assembly
Proteomics of Poxvirus
 Infectious Forms:
1) Intracellular Mature Virions (IMV)
2) Intracellular Enveloped Virus (IEV)
3) Cell-Associated Extracellular Enveloped Virus (EEV)
4) Extracellular Enveloped Virus (EEV)
Recent Classification: IMV = MVs (Mature Virions)
IEV = WVs (Wrapped Virions)
EEV = Evs (Extacellular Virions)
Proteomics of Poxvirus
 Early Studies for Protein Analysis in VACV MVs
 SDS-PAGE
 Combination of SDS-PAGE and N-terminal a.a
sequencing
 Identification of 12 unique virus encoded proteins
(Takahashi et al., 1994)
 Two dimensional gel electrophoresis and a.a sequencing
or immunoprecipitation
 Identification of 12 unique major membrane and core
proteins
(Jensen et al., 1996)
Proteomics of Poxvirus
 10 years later methods:
 1) Gel-free liquid chromatography
Identification of 75 viral proteins and their relative
abundance
(Chung et al., 2006)
-enzymes
-transcription factors
-membrane proteins
-core proteins
- host interacting proteins
A4: most abundant protein in MV particles
(core protein complexes with core protein p4a/4a;
morphogenesis)
Proteomics of Poxvirus
 2) High Performance liquid Chromatography or SDSPAGE in combination with electro-mass spectrometry
(Yoder et al., 2006)
 Identificaton of 63 VACV virion proteins
 Confirmed the presence of most previously identified
proteins
 Some previously identified proteins were not identified
( A2.5, A6, A9, A18, A21, A22, A25, A26, A28, A31, A45,
C6, D7, D13, C5.5, G9, H2, H6, I2 and L5)
Proteomics of Poxvirus
3) Gradient centrifugation with sucrose and cesium
chloride
(Resch et al., 2007)
 Identification of 80 proteins including 69 previously
identified and 11 novel protein
 15 previously reported proteins were not identified
 Determination of 10 most abundant MV proteins:
[major proteins: F17, A3, A4, A10, A17 that perform 80%
of MV protein mass)]
 All these studies highlight:
1)important role that different proteomics
technologies have played in detection of proteins
2) importance of defining virion purity
Replication
 Replication in cytoplasm of host cell.
 other DNA viruses in nucleus
 Use host enzymes for DNA synthesis
 Replication without any host cell enzymes for
DNA synthesis.
 all of the enzymes necessary for DNA
synthesis in virion
 can Replicate DNA but not mature in
enucleated cells
Replication
 attachment - vaccinia
 host cell receptors for epidermal growth
factor (EGF)
 VGF for vaccinia growth factor.
 penetration
 direct penetration of the core
 enters cells via clathrin-
coated pits
 require an acid pH for fusion
to occur
 CAN’T fuse directly with the
plasma membrane.
 taken up by invagination of
clathrin coated pits into
endosomes
Replication
 penetration
 endosomes become
acidified,
 latent fusion activity of
the virus proteins
becomes activated
 virion membrane fuses
with the endosome
membrane
 delivery of the internal
components of the virus
to the cytoplasm
Replication
uncoating
 two stages:
 Removal of the outer membrane as enters the cell
 particle (minus its outer membrane) is
further uncoated
 Within minutes of entry:
 viral mRNA transcripts
 early' genes
 ~50% genome
 protein products complete the uncoating
 nucleocapsid released into the cytoplasm
protein and NA synthesis
 viral factories
 bounded by virally synthesized membranes
 form the envelope of released mature virus
 proteins
 early
 VGF
 secreted
 causes non-infected cells to divide
[proliferative disorders in some poxvirus infections]
protein and NA synthesis
 proteins
 early
 VCP
 binds to C4b
 blocks the activation of classical complement
pathway
 protein that binds to and neutralizes interferon
gamma
 intermediate and late genes
 DNA synthesis
 post-translational processing of viral proteins
 structural proteins
Nucleic Acid synthesis
 starts about 1-2 hrs
 makes - 10,000
copies/cell
 self-priming
 may nick at one or
both ends
 from 3" end only - no
Okazaki fragments
Nucleic Acid
synthesis
 formation of
high m.w.
concatemers
 cleaved and
repaired to
make virus
genomes.
Assembly
 some unknown contribution from the cell
 poxvirus gene expression and genome replication
occur in enucleated cells
 maturation is blocked
 Assembly not understood
 probably involve interactions with the
cytoskeleton
 e.g. actin-binding proteins
Assembly
 Inclusions formed in cytoplasm
 mature into virus particles
 Actin 'comet tails' form
 shoot IEV through the cytoplasm to the cell surface
 possibly into adjacent cells
 an alternative mechanism for cell to cell spread?
 Highly processed and packaged genomic DNA
accumulates
 mature viral particles within 24 hours of infection
Release
 minority of mature enveloped virus fuse with the host cell
plasma membrane
 released from the cell
 responsible for spread of the infection throughout body
 Most remains associated with the cell
VACV
Life
Cycle
AGENTS OF DISEASE
IN
POXVIRUSES
Smallpox
 variola virus (VV) and vaccinia are the best known.
 VV strains are divided into
 variola major (25-30% fatalities)
 variola minor
 same symptoms but less than 1% death rate
 Incubation period is about 12 days
 Initial symptoms include
 high fever
 Fatigue
 head and back aches
 2-3 days later
 lesions appear
 progress from macules to
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papules, and pustular
vesicles.
small blisters that itch and
are extremely painful
begin developing on the
bodies extremities
spread to the rest of body
Twelfth day, the blisters scab
over and leave permanent
pitted scars.
Death usually results if the
virus reaches the brain, heart
or lungs.
Poxvirus Infection
 There is no other reservoir for VV but humans
 VV causes only acute infections, from which
the infected person either:
 a) dies
 b) recovers with life-long immunity
 Vaccinia virus is an effective immunogen.
 first appeared in China and
the Far East at least 2000
years ago.
 The Pharaoh Ramses V
died of smallpox in 1157
B.C.
 skin lesions found on his
mummy
 Marcus Aurelius Antonius,
 Roman
philosopher-emperor,
 another victim;
 During his reign
smallpox wiped out
2,000 people a day.
History
 Chinese healers used
variolation
 dried scabs from
smallpox victims,
 ground to a powder
 blown up the nose.
 worked better if use
variola minor
 widely practiced in
the middle east for
many centuries,
 Turkey
 fluid from smallpox
vesicles scratched
into the recipient's
arm
Vaccination/
Variolization
POX Vaccinaion
 For more than 100 years, the "vaccine strains" were
propagated from arm-to-arm
 Vaccination was almost universally adopted
worldwide around 1800
 for at least last 50 years, Vaccinia has been a distinct
virus from Cowpox
 molecularly most similar to Buffalopox
 United States stopped vaccinating its military in 1989
 civilians in the early 1980s
 Recently have started to vacccinate again
Pox Vaccination
 current VACCINIA VACCINE
 Dryvax: the vaccinia (smallpox) vaccine currently
licensed in the United States
 a lyophilized, live-virus preparation of infectious
vaccinia virus
(Wyeth Laboratories, Inc., Marietta, Pennsylvania).
 Previously:
 calf lymph with a seed virus derived from the New York
City Board of Health (NYCBOH) strain of vaccinia virus
and has a minimum concentration of 108 (PFU)/ml.
 inoculation at other sites
. autoinoculation
 face, eyelid, or other persons
 (~ 6/10,000)
 Erythematous or urticarial rashes
 Side
. Effects and Less Severe
Adverse Reactions
 Reaction at site
 swelling and tenderness of
regional lymph nodes,
 fever
 Approximately 70% of children
experience >1 days of
temperatures >100 F
 15%-20% of children
experience temperatures
>102
 Moderate to Severe
Adverse Reactions
 eczema vaccinatum
 localized or systemic
dissemination of
vaccinia virus
 generalized vaccinia
 vesicular rash
 ~3/10,000 vaccinations
vaccinia necrosum
 severe, potentially fatal
illness
 progressive necrosis in the
area of vaccination
 postvaccinial encephalitis
 15%-25% die
 25% have permanent
neurological disorders
ERRADICATION
 Less than 40 years ago, smallpox was
endemic in 31 countries
 Yugoslavia as late as the early 1970s
 1960's over 2 million people/year die
 WHO in 1965 decided to achieve eradication
 last naturally occurring outbreak was in
Somalia on 26th October 1977
 Endemic smallpox was declared eradicated in
1980 by the (WHO).
ERRADICATION
 possible for 4 reasons:
 single stable serotype
 no other reservoir for variola virus than humans
 Infection spreads only from close contact
with infected persons
 Vaccinia virus is an effective immunogen
ERRADICATION
 variola virus causes only acute infections
 infected person either:
 dies
 recovers with life-long immunity
 most commonly from days 3-6 after onset of fever.
 Thus only infectious after show signs and symptoms
 know who exposed so can isolate
 Strict quarantine with respiratory isolation
 minimum of 16-17 days
 incubation : 10 - 12 days
After eradication
 What to do with existing stocks
 consolidated into two collections
 1976, WHO urged 75 labs in several countries that retained
stocks of variola virus to destroy or transfer them to
official WHO repositories in U.S. and Soviet Union.
 South Africa was last to destroy its virus stocks in 1983
 CDC keeps about 400 different strains
 Moscow laboratory 200 strains in Novizbersk, Russia
 extent of stockpiles in other parts of the world
unknown.