Download 11 M401 Large DNA Virus 2012 - Cal State LA

Replication
of Large
DNA Virus
Herpesvirus,
Poxvirus
Family Herpesviridae
• “creeping” spread of rash & vesicle lesions
• Widely found in nature – plants, fungi,
animals, humans
• Highly infectious
• Infections – acute, persistent, transform
• Eight Human herpesvirus (HHV 1-8)
• Also primate, bovine, equine, swine, murine,
avian herpesvirus
Herpes Simplex Virus
(HSV-1)
• Infect mucous
membranes and skin
• HSV-1: mainly oral &
facial area
• Latent in neurons
Herpes Simplex Virus -2
(HSV-2)
• Mainly genital area
• Most infections are
asymptomatic
• Symptoms of genital
lesions soon after
exposure, last ~10 days
• Latent in neurons, most
have recurrent episodes
within first year
• Mother with active
infection may transmit to
newborn during delivery
Genital Herpes: USA
Epidemic
• Estimate >20 million infections
• Sexually Transmitted Diseases (STD’s)
– Increasing since 1960’s
– Social change (sexual freedom, changing moral
standards, sex outside of marriage)
– Birth control pill developed (non-barrier
contraception)
– Difficult problem for Public Health
Varicella/Zoster Virus
(VZV)
• One virus, two
diseases
• Varicella –
chickenpox
• Latent in neurons
• Zoster – shingles,
uncommon
reactivation along
nerve trunk in
adults
Epstein-Barr Virus (EBV)
• Infectious
mononucleosis
• Infects B lymphocyte,
epithelial, fibroblast
cells
• Latent in lymphoid
tissue
• Co-carcinogen –
Burkitt’s lymphoma,
nasopharyngeal
carcinoma
Human Cytomegalovirus
(HCMV)
• “giant cells” in culture
• syncytia forms multinucleated cell
• Infects monocyte, lymphocyte,
epithelial cell
• Latent in lymphoreticular cells
• USA – leading viral infection of
fetus/newborns
Human Herpesvirus 6
(HHV-6)
• Exanthema subitum (roseola)
• Common rash in young children
• Infects lymphocytes
HHV-7
• Isolated from lymphocytes of AIDS
patient
• “orphan” virus
• No associated disease
HHV-8
• Infects lymphocyte, vascular
endothelial cells
• Viral DNA found in Kaposi’s sarcoma
tissue of AIDS patients
• Co-carcinogen for Kaposi’s sarcoma
HSV-1
• Envelope with surface projections, 200 nm
• Tegument (matrix) structure between
capsid and envelope
• Icosahedral capsid, 130 nm
• Core with virus DNA wound in cylinder
HSV Genome: dsDNA
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Linear, one strand has nicks, 150 kbp
Two unique components (UL, US)
Terminal and internal repeat sequences
Highly conserved “a” sequence at both
ends (used for genome recognition and
insertion into capsid)
HSV Genetic & Transcription Map
HSV: Attachment/
Entry
• Viral surface glycoproteins
• Host cell heparan sulphate
proteoglycans
• Viral attachment blocked
by polycations (polylysine,
neomycin)
• Fusion of viral envelope
with cell plasma membrane
• Capsid into cytoplasm
• Release of VHS (virion
host shut-off) tegument
protein that degrades cell
mRNA in cytoplasm
HSV: Uncoating
• Viral capsid
transported to
nuclear membrane
• Release of DNA into
nucleus
• Viral tegument
protein αTIF (transinducing factor)
transported into
nucleus activates
virus transcription
HSV: mRNA
Transcription
• DNA genome circularizes
• Promoter/enhancer sites activated by viral
αTIF and cell DNA-binding proteins (Oct1, SP1)
• Transcription from both DNA strands,
bidirectional (clockwise, counterclockwise)
• Uses cell RNA pol II
HSV: Regulated Gene
Expression
• Immediate-Early – α gene products,
mainly regulatory
• Early – β gene products, mainly viral
enzymes and proteins for DNA
synthesis
• Late – γ gene products, some
regulatory, mainly structual proteins
Cascade of HSV Transcription
DNA Replication (Rolling Circle)
• Synthesis of DNA in a
long strand (head-totail concatemers)
• Viral enzymes
• Nick DNA strand,
ssDNA rolls off
• Continous and
discontinous (Okazaki
fragments) DNA
replication
• Concatemers later
cleaved into genome
size (recognition of
“a” terminii)
HSV: Assembly
• Viral proteins
transported
into nucleus,
assemble into
capsid
• Viral DNA
“head-full”
insertion into
capsid
HSV: Release
• Viral “primary” tegument
protein associate with viral
glycoprotein, buds through
inner & outer nuclear
membrane, releasing capsid
into cytoplasm
• Capsid migrates to
tegument proteins and
picks up envelope by
budding into exocytic
vesicle
• Virus inside vesicles of
cytoplasm; either remain
cell associated or
“secreted” to outside
Latent Infection
• Virus ascend up sensory
nerve to neuron
• Viral DNA with cell
histones and established
in host cell as “episome”
• Expresses LAT (latencyassociated transcripts)
• No infectious virus
replication
• May be reactivated
(immune suppression,
stress, injury, UV light,
hormone)
HSV
Infection:
Productive
vs
Latent
Reading & Questions
• Chapter 17: Replication of Some
Nuclear-Replicating DNA Viruses
Class Discussion –
Lecture 11a
• 1. How does HSV upon release of its
DNA genome insure that it will be
transcribed?
• 2. Like a good friend, HSV and its
host cell have a lifetime relationship.
How is this possible?
• Viruses of vertebrates
and insects
• Large “brick” shape,
200x300 nm
– External, inner envelope
– Lateral bodies
– Complex coat of tubular
structures
• Replication occurs in
cytoplasm
• Benign tumors in
experimental hosts
Family Poxviridae
Human Poxviruses
• Characteristic rash and
“pocks”
• Variola – smallpox
– Transmitted by
inhalation
and infects respiratory
tract, systemic
infection
– eradicated by WHO
vaccination (1977)
• Vaccinia – “cowpox” lab
recombinant used for
vaccine
• Molluscum contagiosum
– localized lesions,
transmitted by contact
Occassional Poxvirus
Zoonosis to Humans
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Localized lesions
Transmitted by contact
Orf – sheep, goat
Cowpox – rodents, cats, cows
Monkeypox – monkeys, squirrels
Vaccinia Virus Genome: dsDNA
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Linear, 186 kbp
Covalently closed ends (“hairpin” loops)
Inverted terminal repeats (10 kbp)
Conserved central region
Genes code for enzymes needed for
RNA/DNA synthesis
Vaccinia Virus:
Entry/Uncoating
• Fusion of virus with plasma membrane
or entry by endocytosis
• Release of viral core into cytoplasm
• Viral proteins shut off host functions
• Further uncoating leads to “early”
viral transcription/proteins in
cytoplasm
Vaccinia Virus: Expression
of “Early” Genes
• Virus core brings in enzymes required
for viral transcription
• Half of genome is expressed from
“early” gene promoters (activated by
viral DNA binding proteins)
• Express enzymes needed for DNA
replication
DNA Replication
• Occurs in cytoplasm
• Nick at end creates a
free 3’ OH, self-priming
• DNA synthesis displaces
parent strand, two
genome concatemer
(tail-to-tail)
• Continued DNA
synthesis displaces two
genome strand
concatemer (tail-to-tail,
head-to-head)
• Cleaved into two genome
lengths
• Fill in and ligate ends
into dsDNA, closed ends
Vaccinia Virus: Expression
of “Late” Genes
• Switch due to viral regulatory
proteins and configuration of newly
replicated viral DNA
• Use of “late” promoters
• Expression of some enzymes, mainly
structual proteins
Vaccinia Virus: Assembly
and Release
• Sequential developmental stages in
cytoplasm
• Viral membrane form crescent and circular
structures
• Nucleoprotein mass forms with immature
envelope and buds through golgi membrane
for envelope
• Release by budding through plasma
membrane
Vaccinia Virus Replication Cycle
Recombinant
Vaccine
• Poxviruses have high
recombination rate
• Dual infection of
vaccinia virus +
recombinant plasmid
cloning vector with
foreign virus gene
• Use of recombinant
vaccinia virus + foreign
gene for possible
protective vaccine
Smallpox Virus: Potential
Terrororist Weapon?
• Susceptible population
• Easily transmitted by inhalation
• Highly virulent strains (up to 40%
mortality)
• Smallpox virus stored in two Public Health
Labs (USA, former Soviet Union)
• Fear?
• Best defense?
Reading
• Chapter 18: Replication of
Cytoplasmic DNA Viruses
Class Discussion –
Lecture 11b
• 1. What would you postulate for the origin
of Poxviruses?
• 2. If a terrorist ask your suggestion for a
biological agent, would you tell him to go
buy a herpesvirus or a smallpox virus from
an underpaid government research
microbiologist ?
• 3. Since smallpox has now been
eradicated, would it be a good or bad ideal
to destroy the remaining virus samples in
the U.S.?
MICR 401 Final Exam
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Tuesday, Dec. 4, 2012
1:30 – 3:00pm
Papovavirus thru Hepadnavirus
Case Study and Questions #9-15
Lecture & Class Discussion Questions,
Reading & Chapter Questions
• Exam:
– Objective Questions (MC, T/F, ID)
– Short Essay Questions