| Poxviruses |
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A model of a poxvirus cut-away in
cross-section to show the internal
structures. Poxviruses are shaped like
flattened capsules/barrels or are lens or
pill-shaped. Their structure is complex,
neither icosahedral nor helical. This
model is based on Vaccinia, the smallpox
virus. The structures are also highly
variable and often incompletely studied.
cross-section to show the internal
structures. Poxviruses are shaped like
flattened capsules/barrels or are lens or
pill-shaped. Their structure is complex,
neither icosahedral nor helical. This
model is based on Vaccinia, the smallpox
virus. The structures are also highly
variable and often incompletely studied.
Tech-level: 2/3 (advanced)
Prerequisites: ribosomes, nucleus, cell structure, cell membranes
Poxviruses are very large for viruses, and are complex. The outer envelope of the virus consists of two layers: the
thicker outer layer containing surface tubules or short filaments of helically wound protein subunits (and typically
with a hollow channel running through the centre of each tubule) and the thinner inner layer (which is possibly a
lipid bilayer - it is known that part of the envelope consists of a lipid bilayer membrane). The surface tubules are
short in Vaccinia (an Orthopoxvirus), but in Orf virus (a Parapoxvirus) they form continuous helices that wind
around the virus. In some poxviruses the tubules are replaced by globular protein structures. In Variola an
Orthopoxvirus), the tubules are anchored in the outer envelope layer at one end and project from the virus surface
as flexible virovilli (these have also been reported for Vaccinia).
Inside the envelope is the core and two lateral bodies. The core consists of the core wall - an outer thick palisade
layer, 17 nm thick (so called because it is striated, giving the appearance of many tiny columns) and the thinner,
smooth inner layer or core membrane, 8 nm thick. Inside the core wall, the core is packed with fibrillar material (the
core fibrils) which surrounds the nucleoprotein coil. The nucleoprotein is a continuous helix/cylinder which traverses
the length of the virus three times (forming the three so-called 'triplet elements' seen in cross-section) such that
three segments run parallel to one-another, but the whole is a continuous tube about 250 nm long).
The nucleoprotein consists of the tightly-packed linear double-stranded DNA (dsDNA) genome coated by at least 4
different types of proteins, maintaining the DNA in a superhelical state. A DNA-dependent RNA polymerase is also
present in the nucleoprotein or core. This structure is the helical part of the virus, and suggests that poxviruses
may have evolved from helical viruses by the addition of extra structures. The poxvirus genome is large, 150 to 300
kbp (kbp = thousand base-pairs), contains no introns (see Adenovirus) and contains more than 150 open reading
frames that do not overlap (ORFs - regions of the DNA that are transcribed and so code for potential 'structural'
genes, that is genes other than those that control other genes). This is a lot of genes for a virus! The genome
ends are palindromic tandem repeats (meaning that they consist of short sequences of bases repeated several
times which read the same forwards and backwards, e.g. ...ATATA...).
The whole structure depicted above may be enclosed in a cell envelope - a bilayer phospholipid membrane
acquired from the host cell, with embedded globular viral glycoproteins. (See escape from the host cell below).
Smallpox
The most famous poxvirus is the smallpox virus. (Chicken pox is not caused by a true poxvirus, but rather by
Varicella, or varicella zoster virus (VZV), a member of the herpesvirus family). If you are searching for real-life
monsters, then search no further! Smallpox was truly a terrible disease that caused tremendous misery throughout
the history of humanity, until its recent apparent eradication. Smallpox is infectious, and contracted by prolonged
face-to-face contact with an infected person or by contact with their body fluids, contaminated objects and
sometimes by inhalation. Initially it infects the throat and lymph glands and eventually enters the bloodstream
(viraemia) and attacks bone, spleen and skin cells and causes a characteristic rash of blisters and about one-third
of those who experience symptoms die. It is thought that Pharoah Ramses V of Egypt suffered from smallpox and
possibly died from it, around 1145 BC. Vaccination appears to have eradicated the virus from nature, from 1979
onwards. However, the virus remains in specialised laboratory facilities and the last recorded mortality was in 1978,
in the UK University of Birmingham Medical School, in which two people were accidentally infected from laboratory
stocks, one of whom dies and leading to the principal investigator committing suicide - all very tragic indeed.
Adhesion and Entry
Poxviruses have wide host ranges. Vaccinia produces three types of infectious virions: intracellular mature virus
(IMVs) with the structure shown in the model above, and extracellular enveloped virus (EEV), with an
additional envelope consisting of a phospholipid bilayer membrane with embedded virus proteins and intracellular
enveloped virus (IEV) which is an IMV with an additional double-membrane envelope (consiting of two
phospholipd-bilayer membranes derived from the host cell). These three types of virions have different entry routes
into the host cell, though all are infectious. As they have different outer envelopes, with different viral proteins, they
likely attach to different receptors on the target cell surface membrane. The IMVs are the most common type.
In EEVs, the virus attaches to receptors on the surface-membrane of the target cell, triggering fusion of the virus
and host-cell membranes and release of the virus core into the cytoplasm, as the outer membrane (envelope?) of
the virus is shed on entry to the cell. The core enters the host-cell cytoplasm.
In IMVs, the virus protein A27L binds heparin sulphate, which is a glycosaminoglycan (GAG, or
mucopolysaccharide) side chain of cell surface proteoglycans. GAGs are chains of sugar molecules, made-up of
repeating two-sugar (disaccharide) units linked to a hexosamine (a 6-carbon sugar molecule containing a nitrogen
atom). A proteoglycan is a protein with a GAG linked to it (by a covalent bond). Proteoglycans are major
components of the extracellular matrix (the packing material between cells) and the slime-coat of animal cells.
Additionally in IMVs, the D8L viral protein on the IMV binds chondroitin sulphate (a sulphur-containing GAG).
Early phase (30 minutes after cell infection)
As the core uncoats (revealing the naked DNA which enters the host cytosol), the early genes (those transcribed
and translated, or expressed, early in infection) are transcribed in the host cytoplasm by the viral DNA-dependent
RNA polymerase which the core carried with it. DNA replication produces about 10 000 copies of the viral genome
per host cell! The surface tubules are released inside the cytoplasm and these switch-off the synthesis of cell
proteins by the host cell, which is to commandeered to make viral proteins instead! The products of translation of
some of the early mRNA (messenger RNA) function to disrupt host cell defenses. The viral mRNA is not spliced and
viral enzymes add 5' caps and 3' polyadenyl tails to produce mature mRNA from the primary transcripts. These
early transcripts are produced inside the viral core as it gradually uncoats.
Intermediate phase (100 minutes after cell infection)
The intermediate genes are switched-on, triggering replication of the viral DNA genome. Viral synthesis occurs
inside virally-induced double-membrane bound vesicles (derived from the host endoplasmic reticulum) called
virosomes. DNA replication occurs inside these virosomes, which later disassemble prior to virion assembly in the
late phase.
Although the genome is linear DNA, the ends are covalently closed, forming a circular structure. The viral genomic
DNA is nicked at one end by the enzyme helicase and the DNA begins to unzip into two single-strands and the free
ends fold-over to produce self-priming hairpin loops. (DNA polymerase requires a short double-stranded sequence
to begin synthesis). This and the following steps in DNA replication are illustrated below (click images to enlarge).
To understand the diagram below, recall that:
Prerequisites: ribosomes, nucleus, cell structure, cell membranes
Poxviruses are very large for viruses, and are complex. The outer envelope of the virus consists of two layers: the
thicker outer layer containing surface tubules or short filaments of helically wound protein subunits (and typically
with a hollow channel running through the centre of each tubule) and the thinner inner layer (which is possibly a
lipid bilayer - it is known that part of the envelope consists of a lipid bilayer membrane). The surface tubules are
short in Vaccinia (an Orthopoxvirus), but in Orf virus (a Parapoxvirus) they form continuous helices that wind
around the virus. In some poxviruses the tubules are replaced by globular protein structures. In Variola an
Orthopoxvirus), the tubules are anchored in the outer envelope layer at one end and project from the virus surface
as flexible virovilli (these have also been reported for Vaccinia).
Inside the envelope is the core and two lateral bodies. The core consists of the core wall - an outer thick palisade
layer, 17 nm thick (so called because it is striated, giving the appearance of many tiny columns) and the thinner,
smooth inner layer or core membrane, 8 nm thick. Inside the core wall, the core is packed with fibrillar material (the
core fibrils) which surrounds the nucleoprotein coil. The nucleoprotein is a continuous helix/cylinder which traverses
the length of the virus three times (forming the three so-called 'triplet elements' seen in cross-section) such that
three segments run parallel to one-another, but the whole is a continuous tube about 250 nm long).
The nucleoprotein consists of the tightly-packed linear double-stranded DNA (dsDNA) genome coated by at least 4
different types of proteins, maintaining the DNA in a superhelical state. A DNA-dependent RNA polymerase is also
present in the nucleoprotein or core. This structure is the helical part of the virus, and suggests that poxviruses
may have evolved from helical viruses by the addition of extra structures. The poxvirus genome is large, 150 to 300
kbp (kbp = thousand base-pairs), contains no introns (see Adenovirus) and contains more than 150 open reading
frames that do not overlap (ORFs - regions of the DNA that are transcribed and so code for potential 'structural'
genes, that is genes other than those that control other genes). This is a lot of genes for a virus! The genome
ends are palindromic tandem repeats (meaning that they consist of short sequences of bases repeated several
times which read the same forwards and backwards, e.g. ...ATATA...).
The whole structure depicted above may be enclosed in a cell envelope - a bilayer phospholipid membrane
acquired from the host cell, with embedded globular viral glycoproteins. (See escape from the host cell below).
Smallpox
The most famous poxvirus is the smallpox virus. (Chicken pox is not caused by a true poxvirus, but rather by
Varicella, or varicella zoster virus (VZV), a member of the herpesvirus family). If you are searching for real-life
monsters, then search no further! Smallpox was truly a terrible disease that caused tremendous misery throughout
the history of humanity, until its recent apparent eradication. Smallpox is infectious, and contracted by prolonged
face-to-face contact with an infected person or by contact with their body fluids, contaminated objects and
sometimes by inhalation. Initially it infects the throat and lymph glands and eventually enters the bloodstream
(viraemia) and attacks bone, spleen and skin cells and causes a characteristic rash of blisters and about one-third
of those who experience symptoms die. It is thought that Pharoah Ramses V of Egypt suffered from smallpox and
possibly died from it, around 1145 BC. Vaccination appears to have eradicated the virus from nature, from 1979
onwards. However, the virus remains in specialised laboratory facilities and the last recorded mortality was in 1978,
in the UK University of Birmingham Medical School, in which two people were accidentally infected from laboratory
stocks, one of whom dies and leading to the principal investigator committing suicide - all very tragic indeed.
Adhesion and Entry
Poxviruses have wide host ranges. Vaccinia produces three types of infectious virions: intracellular mature virus
(IMVs) with the structure shown in the model above, and extracellular enveloped virus (EEV), with an
additional envelope consisting of a phospholipid bilayer membrane with embedded virus proteins and intracellular
enveloped virus (IEV) which is an IMV with an additional double-membrane envelope (consiting of two
phospholipd-bilayer membranes derived from the host cell). These three types of virions have different entry routes
into the host cell, though all are infectious. As they have different outer envelopes, with different viral proteins, they
likely attach to different receptors on the target cell surface membrane. The IMVs are the most common type.
In EEVs, the virus attaches to receptors on the surface-membrane of the target cell, triggering fusion of the virus
and host-cell membranes and release of the virus core into the cytoplasm, as the outer membrane (envelope?) of
the virus is shed on entry to the cell. The core enters the host-cell cytoplasm.
In IMVs, the virus protein A27L binds heparin sulphate, which is a glycosaminoglycan (GAG, or
mucopolysaccharide) side chain of cell surface proteoglycans. GAGs are chains of sugar molecules, made-up of
repeating two-sugar (disaccharide) units linked to a hexosamine (a 6-carbon sugar molecule containing a nitrogen
atom). A proteoglycan is a protein with a GAG linked to it (by a covalent bond). Proteoglycans are major
components of the extracellular matrix (the packing material between cells) and the slime-coat of animal cells.
Additionally in IMVs, the D8L viral protein on the IMV binds chondroitin sulphate (a sulphur-containing GAG).
Early phase (30 minutes after cell infection)
As the core uncoats (revealing the naked DNA which enters the host cytosol), the early genes (those transcribed
and translated, or expressed, early in infection) are transcribed in the host cytoplasm by the viral DNA-dependent
RNA polymerase which the core carried with it. DNA replication produces about 10 000 copies of the viral genome
per host cell! The surface tubules are released inside the cytoplasm and these switch-off the synthesis of cell
proteins by the host cell, which is to commandeered to make viral proteins instead! The products of translation of
some of the early mRNA (messenger RNA) function to disrupt host cell defenses. The viral mRNA is not spliced and
viral enzymes add 5' caps and 3' polyadenyl tails to produce mature mRNA from the primary transcripts. These
early transcripts are produced inside the viral core as it gradually uncoats.
Intermediate phase (100 minutes after cell infection)
The intermediate genes are switched-on, triggering replication of the viral DNA genome. Viral synthesis occurs
inside virally-induced double-membrane bound vesicles (derived from the host endoplasmic reticulum) called
virosomes. DNA replication occurs inside these virosomes, which later disassemble prior to virion assembly in the
late phase.
Although the genome is linear DNA, the ends are covalently closed, forming a circular structure. The viral genomic
DNA is nicked at one end by the enzyme helicase and the DNA begins to unzip into two single-strands and the free
ends fold-over to produce self-priming hairpin loops. (DNA polymerase requires a short double-stranded sequence
to begin synthesis). This and the following steps in DNA replication are illustrated below (click images to enlarge).
To understand the diagram below, recall that:
- Each of the two single-strands in the DNA duplex (double helix) has chemically distinct ends, which we label
3’ and 5’. - Complementary strands hydrogen-bond to form the duplex, with the hydrogen bonds forming between
complementary bases with A pairing with T and c with G. - The 3’ end of one strand is opposite the 5’ end of the other complementary strand.
- DNA polymerase (DNA-dependent DNA polymerase) reads the template strand in the direction 3' to 5‘ and so
synthesises a new complementary strand which grows in the 5’ to 3’ direction.
Selected References
Che-Sheng Chung, Jye-Chian Hsiao, Yuan-Shau Chang & Wen Chang, 1998. A27L Protein Mediates Vaccinia
Virus Interaction with Cell Surface Heparan Sulfate. J. Virology, Feb 1998: 1577–1585.
Jye-Chian Hsiao, Che-Sheng Chung & Wen Chang, 1999. Vaccinia Virus Envelope D8L Protein Binds to Cell
Surface Chondroitin Sulfate and Mediates the Adsorption of Intracellular Mature Virions to Cells. J. Virology Oct.
1999: 8750–8761.
Paula Traktman, 1996. Poxvirus DNA Replication. DNA Replication in Eukaryotic Cells, Cold Spring Harbor
Laboratory Press.
Che-Sheng Chung, Jye-Chian Hsiao, Yuan-Shau Chang & Wen Chang, 1998. A27L Protein Mediates Vaccinia
Virus Interaction with Cell Surface Heparan Sulfate. J. Virology, Feb 1998: 1577–1585.
Jye-Chian Hsiao, Che-Sheng Chung & Wen Chang, 1999. Vaccinia Virus Envelope D8L Protein Binds to Cell
Surface Chondroitin Sulfate and Mediates the Adsorption of Intracellular Mature Virions to Cells. J. Virology Oct.
1999: 8750–8761.
Paula Traktman, 1996. Poxvirus DNA Replication. DNA Replication in Eukaryotic Cells, Cold Spring Harbor
Laboratory Press.
Late phase (140 min to 48 hours after cell infection)
Towards the end of the viral replication-cycle, the late genes are activated. These produce the structural proteins
that go to make-up the assembled virus particles (virions).Initially immature particles are produced which then
mature into brick-shaped intracellular mature virion (IMV), similar to that in our model above.
Escape from the Host Cell
IMV virion can be released upon cell lysis, and is infectious. This is the most abundant form of the mature virus.
Alternatively, the virions can become enveloped by a double membrane (two phospholipid-bilayer membranes)
derived from the host cell's Golgi apparatus, forming an intracellular enveloped virus (IEV). These virions are
delivered to the host cell-surface membrane, by using the host cell's cytoskeleton of microtubules and actin
filaments which form behind the virion in an actin-tail which propels the virus to the host-cell surface, where the
outer membrane fuses with the host cell-membrane and releases a mature virion with a single membrane envelope
(a phosopholipid bilayer with embedded viral proteins). This later type of infectious particle is called an external
enveloped virion (EEV).
Towards the end of the viral replication-cycle, the late genes are activated. These produce the structural proteins
that go to make-up the assembled virus particles (virions).Initially immature particles are produced which then
mature into brick-shaped intracellular mature virion (IMV), similar to that in our model above.
Escape from the Host Cell
IMV virion can be released upon cell lysis, and is infectious. This is the most abundant form of the mature virus.
Alternatively, the virions can become enveloped by a double membrane (two phospholipid-bilayer membranes)
derived from the host cell's Golgi apparatus, forming an intracellular enveloped virus (IEV). These virions are
delivered to the host cell-surface membrane, by using the host cell's cytoskeleton of microtubules and actin
filaments which form behind the virion in an actin-tail which propels the virus to the host-cell surface, where the
outer membrane fuses with the host cell-membrane and releases a mature virion with a single membrane envelope
(a phosopholipid bilayer with embedded viral proteins). This later type of infectious particle is called an external
enveloped virion (EEV).
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