Pox Viruses

1
Pox Viruses

• 2 subfamilies
• Chordopoxviruses (vertebrate viruses)
• 2 human viruses variola (smallpox) and
  molluscum contagiosum
• vaccinia the most widely studied pox virus
• - natural host not known (infects humans and
  many other species)
• - genome is 90 identical to variola
• - used as smallpox vaccine for approx. 100
  years
• 2) Entomopoxviruses (insect viruses)

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Virions

• Rounded rectangles (brick-shaped) 350 x 270 nm
• Dumbbell-shaped core (brick-shaped at other
  angle) surrounded by an envelope and 1 or 2
  additional membranes

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Virions

• Two type of infections virions
• Intracellular mature virions (IMV)
• - 1 membrane in addition to core envelope
• (2 membranes in total)
• - more stable than EEV outside the host
• - involved in spread from 1 host to the next
• 2) Extracellular enveloped virions (EEV)
• - 2 membranes in addition to core envelope
• (3 membranes in total)
• - required for efficient cell to cell spread
  within
• the host
• Core
• - Contains genome and many viral proteins
  including RNA polymerase and other proteins
  required for synthesizing, polyadenylating,
  capping and methylating mRNA

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Genome

• 2 DNA strands connected by a hairpin loop to form
  a continuous, mostly double-stranded molecule
• 130-280 kbp
• Encoded approx. 200-250 proteins
• Ends contain inverted terminal repeats

5
Viral Entry

• No specific cell receptor known for any poxvirus
• Virus can bind and enter any cell but not all
  cells are permissive for viral replication
• three envelope proteins thought to mediate
  attachment to cell surface
• IMV outer membrane fuses to plasma membrane
• EEV- taken up by endocytosis
• - outer membrane ruptures releasing IMV
• - IMV membrane then fuses with vesicle
  membrane
• In the cytoplasm, a second uncoating step occurs
  so that the nucleoprotein complex is released
  from the core
• Cores are then transported along microtubules to
  regions near ER where DNA is released from core.

6
Gene Expression

• Early, intermediate and late gene classes
  (expressed in cytoplasm)
• No splicing (pox does not encode the splicing
  machinery)
• Early Genes
• Encode proteins involved in intermediate gene
  expression, host interactions and DNA replication
• Each gene has conserved promoter sequence
• Expression is induced by viral proteins in the
  virion core
• ie. RNA polymerase, RAP94, VETF (vaccinia early
  transcription factor)
• - RAP94 and VETF bind the RNA polymerase and are
  required to activate early promoters (VETF binds
  sequences in early promoters)
• -other virion components are needed for
  transcription termination, capping and
  polyadenylation

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Gene Expression

• Intermediate Genes (5 in vaccinia)
• Expressed after DNA replication
• Have different promoter sequences than early
  genes
• Intermediate proteins activate late gene
  expression
• Late Genes
• Expressed after intermediate genes
• Have different promoter sequences than early or
  intermediate genes
• Virion components including proteins required for
  early gene transcription (eg. RAP94 and VETF)
• Both Intermediate and late mRNAs have
• 5polyA heads - 30-50 As upstream of AUG
  translation start site due to slippage during
  transcription by viral RNA polymerase (may help
  in translation)
• Heterogeneous 3 ends since transcription does
  not terminate at 1 precise site

8
DNA Replication

• Occurs at viral factories which are surrounded
  by membranes
• Rough ER membranes wrap around the viral genomes
  just prior to DNA replication to form
  mini-nuclei
• Subsequent transcription and translation also
  occurs with the viral factories which are then
  disassembled prior to virion formation.
• Vaccinia encodes several enzymes for generating
  the nucleotides needed for DNA replication
• Also encodes the proteins needed for DNA
  synthesis including DNA polymerase and its
  processivity factor, ssDNA binding protein, DNA
  ligase
• Approx 10,000 viral genomes produced per cell

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Vaccinia DNA Replication

• Starts by nicking DNA near the end of the genome
  (exact position and protein responsible are not
  known)
• 3OH at nick serves as primer
• Replication occurs continuously by strand
  displacement (rolling hairpin mechanism like
  parvovirus)
• Produces tail to tail dimers which can undergo
  further replication to produce higher-order
  concatemers
• Dimers and concatemers are resolved into
  individual genomes during late-stage
  transcription (involves unknown viral nuclease
  and DNA ligase)

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Vaccinia DNA Replication
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Virion Production

• Occurs in the cytoplasm
• Immature virions (IV) are assembled first. These
  have 2 membranes that come from the Golgi by a
  wrapping mechanism.
• IVs then mature from a spherical shape to the
  brick-shaped IMV (intracellular mature virion)
• IMVs are released from the cell if the cell lyses
• IMVs can acquire 2 additional membranes from the
  Golgi to form intracellular enveloped virions
  (IEV)
• IEVs are transported to the plasma membrane,
  where the outer membrane fuses to the plasma
  membrane releasing the EEVs (extracellular
  enveloped virions) from the cell
• EEVs that remain associated with the cell surface
  are referred to as CEVs (cell-associated
  enveloped virion)
• CEVs are important for cell to cell transmission
• EEVs are important for longer range spread of the
  virus within the host

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Vaccinia Egress
13
Vaccinia Egress
TGN trans golgi network
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Actin Tails
Actin (green) in vaccinia (red) infected cell
Actin (green) in uninfected cell

• CEVs induce the formation of actin tails beneath
  the plasma membrane where CEV is attached
• Actin tails push the CEV from the cell surface
  and into neighbouring cells

Actin tail projections (green) on surface of
vaccinia (red) infected cell
Electron micrograph of CEV and actin tail
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Summary of Vaccinia Infection
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Effects on Host Cell

• Has profound cytopathic effects on the cell and
  has several mechanisms to combat host immune
  responses
• Immediate inhibition of host DNA synthesis
• Immediate inhibition of host protein synthesis
  due to
• Rapid degradation of some host mRNA (by viral
  protein that cleaves off mRNA caps decapping
  enzyme)
• Inhibition of host mRNA synthesis and processing
• 3) Inhibition of anti-viral interferon response
• - two different proteins act on PKR pathway so
  translation continues -1 is homologue of
  cellular eIF-2a (inhibits phosphorylation of
  eIF2a by PKR) the other sequesters dsRNA to
  inhibit PKR activation
• 4) Inhibition of cytokine signalling
• - involves several viral protein that mimic
  cellular proteins in cytokine pathway
• 5) Inhibition of complement system
• - vaccinia makes a protein that is secreted and
  inhibits complement-based immune recognition

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Clinical Aspects

• Vaccinia and cowpox
• usually only cause a lesion at the site of
  introduction in the skin
• both have been used as smallpox vaccine (usually
  live vaccinia virus is used)
• Vaccinia virus infection can be life-threatening
  in people with eczema (eczema vaccinatum)
• Molluscum Contagiosum
• - common in children
• - pimple-like bumps (papules) on the skin that
  eventually resolve on their own
• - can be sexually transmitted in adults
• - bigger problem in immunocompromised patients
• Monkey Pox
• Natural hosts are ground squirrels (can also
  infect monkeys)
• Can infected humans and cause disease similar to
  smallpox (can be lethal)
• Little human to human spread

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Smallpox

• Plagued humans for over 3000 years
• Eradicated (last case in 1977 in Somalia)
• Immunization stopped in 1980
• New stocks of vaccine have recently been made due
  to bioterrorism threat (vaccine works even if
  given 3-4 days after infection)
• Enters through respiratory tract then spreads
  through the body
• There is a 1-2 week incubation period with no
  symptoms and no virus produced, where the virus
  amplifies
• then a 4 day period of systemic disease with high
  fever, headache and vomiting
• then a 12 day period with fever and a rash over
  the entire body (and internal passages)
• Outcome was either death (25-30) or recovery
  with elimination of virus and prolonged immunity
  to variola
• Survivors were left with permanent scars and were
  often blind

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Smallpox Rash
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Giant Viruses

• Herpes and Pox viruses are the largest viruses
  that infect human cells but there are larger
  viruses that infect other organisms (Giant
  Viruses)
• The largest known virus is the Mimivirus which
  infects amoebae
• Mimivirus genome 1,200 Kbp (dsDNA)
• - encodes about 1262 proteins including protein
  translation factors
• Larger than the genome of many prokaryotic cells
• Replicates in the cytoplasm in a giant viral
  factory (similar in size to the nucleus)

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Top-20 virus genome sizes
Name Genome size (kb) Family
Acanthamoeba Polyphaga Mimivirus 1181.4
Mimiviridae Bacillus phage G 497.5
Myoviridae Coccolithovirus EhV-86 407.3
Phycodnaviridae Paramecium bursaria Chlorella
virus NY-2A 368.7 Phycodnaviridae Canarypox
virus 359.9 Chordopoxvirinae Paramecium
bursaria Chlorella virus AR158 344.7
Phycodnaviridae Ectocarpus siliculosus virus
335.6 Phycodnaviridae Paramecium bursaria
Chlorella virus 1 330.7 Phycodnaviridae
Paramecium bursaria Chlorella virus MT325
314.3 Phycodnaviridae Shrimp white spot
syndrome virus 305.1 Nimaviridae Fowlpox
virus 288.5 Chordopoxvirus Pseudomonas
phage phiKZ 280.3 Myoviridae Bacteriophage
KVP40 244.8 Myoviridae Pongine herpesvirus
4 241.1 Herpesviridae Melanoplus
sanguinipes entomopoxvirus 236.1
Entomopoxvirus Human herpesvirus 5 (CMV)
strain Merlin 235.6 Herpesviridae
Bacteriophage Aehl 233.2 Myoviridae
Amsacta moorei entomopoxvirus 232.4
Entomopoxvirus Human herpesvirus 5 (CMV)
strain AD169 230.3 Herpesviridae Murid
herpesvirus 1 230.3 Herpesviridae
From Claverie et al 2006 Virus Research 117, 133