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Assembly, Maturation, and Release
Assembly, Maturation, and Release
(Getting it all together and leaving!)
(Getting it all together and leaving!)
AssemblyAssembly
The final phase of the viral life cycle is FUNDAMENTALLY DIFFERENT than that of any other type of organism.
Viruses are assembled from component parts, not from division of a pre-existing virus.
For naked viruses: Spontaneous self assembly can occur in vitro by
combining pre-formed component parts. Assembly may require specific virus encoded,
nonstructural proteins. The particle may be assembled from precursor
proteins that are subsequently modified to form the infectious virion.
The final phase of the viral life cycle is FUNDAMENTALLY DIFFERENT than that of any other type of organism.
Viruses are assembled from component parts, not from division of a pre-existing virus.
For naked viruses: Spontaneous self assembly can occur in vitro by
combining pre-formed component parts. Assembly may require specific virus encoded,
nonstructural proteins. The particle may be assembled from precursor
proteins that are subsequently modified to form the infectious virion.
AssemblyAssembly
Enveloped viruses can’t assemble in vitro because their envelope is derived from a host cell membrane.
Assembly of naked or enveloped viruses always requires protein-protein interactions and protein-nucleic acid interactions. The order of assembly could occur in 2 different ways: The genomic nucleic acid serves as a focus for
assembly of the capsid surrounding it. A hollow capsid is formed and is then filled with
the genomic nucleic acid.
Enveloped viruses can’t assemble in vitro because their envelope is derived from a host cell membrane.
Assembly of naked or enveloped viruses always requires protein-protein interactions and protein-nucleic acid interactions. The order of assembly could occur in 2 different ways: The genomic nucleic acid serves as a focus for
assembly of the capsid surrounding it. A hollow capsid is formed and is then filled with
the genomic nucleic acid.
AssemblyAssembly
The choice of which strategy to use is a function of the capsid architecture Helical viruses use the first strategy Icosahedral viruses use the second strategy
Rigid Helical viruses (Tobacco mosaic virus) Composed of RNA plus identical capsomers
arranged in a helix surrounding it. TMV capsid proteins only recognize TMV
RNA. This means that the protein-nucleic acid interactions are very specific.
The choice of which strategy to use is a function of the capsid architecture Helical viruses use the first strategy Icosahedral viruses use the second strategy
Rigid Helical viruses (Tobacco mosaic virus) Composed of RNA plus identical capsomers
arranged in a helix surrounding it. TMV capsid proteins only recognize TMV
RNA. This means that the protein-nucleic acid interactions are very specific.
TMVTMV
TMV assemblyTMV assembly
First, 34 capsid proteins assemble into a pair of disks.
The outer portions interact to hold the two disks together, while the inner portion has a gap where RNA binds.
When the RNA enters, the gap is closed to hold the RNA in place.
First, 34 capsid proteins assemble into a pair of disks.
The outer portions interact to hold the two disks together, while the inner portion has a gap where RNA binds.
When the RNA enters, the gap is closed to hold the RNA in place.
TMV assemblyTMV assembly
Simple assembly modelSimple assembly model
TMV assemblyTMV assembly
RNA interacts with the disks beginning at the “pac site” which is about 1000 bases from the 3’ end of the genome.
The pac site consists of ~ 500 bases that can form a series of hairpin loops.
Loop 1 contains the active residues (GGG) which are highlighted on the following picture.
RNA interacts with the disks beginning at the “pac site” which is about 1000 bases from the 3’ end of the genome.
The pac site consists of ~ 500 bases that can form a series of hairpin loops.
Loop 1 contains the active residues (GGG) which are highlighted on the following picture.
TMV assemblyTMV assembly
TMV assemblyTMV assembly
Pac sequence loop 1 enters the disks and becomes intercalated into the gap.
When the flexible residues close upon the RNA, the disks shift their conformation to a lock-washer arrangement. This is the beginning of the helical conformation.
Assembly proceeds in the 5’ to 3’ direction as RNA is drawn up through the hole in the helix and intercalated into additional disks as they are added.
Pac sequence loop 1 enters the disks and becomes intercalated into the gap.
When the flexible residues close upon the RNA, the disks shift their conformation to a lock-washer arrangement. This is the beginning of the helical conformation.
Assembly proceeds in the 5’ to 3’ direction as RNA is drawn up through the hole in the helix and intercalated into additional disks as they are added.
TMV assemblyTMV assembly
AssemblyAssembly
Flexible helical capsids The helical nucleocapsids of enveloped viruses are
flexible. Since the virus has an envelope to shield the
nucleic acid from the elements (environment), the capsids don’t have the job of shielding the nucleic acid.
Therefore, they are organized in a looser arrangement and the RNA may actually be wrapped around the outside of the nucleocapsid.
The 5’ ends of the genomic RNA may have pac-like regions to assure that only the correct RNA is assembled in the nucleocapsid.
Flexible helical capsids The helical nucleocapsids of enveloped viruses are
flexible. Since the virus has an envelope to shield the
nucleic acid from the elements (environment), the capsids don’t have the job of shielding the nucleic acid.
Therefore, they are organized in a looser arrangement and the RNA may actually be wrapped around the outside of the nucleocapsid.
The 5’ ends of the genomic RNA may have pac-like regions to assure that only the correct RNA is assembled in the nucleocapsid.
AssemblyAssembly
Icosahedral RNA viruses Remember that an icosahedron has 20
faces and each face is composed of 3 subunits (or multiples of 3). The subunits may be identical or different. There are also 12 vertices or corners.
All vertices are surrounded by 5 identical capsomers (pentamer) and the intersections between faces are formed by six capsomers (hexamers)
Icosahedral RNA viruses Remember that an icosahedron has 20
faces and each face is composed of 3 subunits (or multiples of 3). The subunits may be identical or different. There are also 12 vertices or corners.
All vertices are surrounded by 5 identical capsomers (pentamer) and the intersections between faces are formed by six capsomers (hexamers)
Icosahedral virusesIcosahedral viruses
Icosahedral RNA virusesIcosahedral RNA viruses
The capsomer proteins all appear to share common structural themes which are related to their roles in assembly: Eight beta sheets linked together by
alpha helices or random coils An arm of variable length is found at the
amino terminus The tertiary structure looks like a cheese
wedge with an arm extending away.
The capsomer proteins all appear to share common structural themes which are related to their roles in assembly: Eight beta sheets linked together by
alpha helices or random coils An arm of variable length is found at the
amino terminus The tertiary structure looks like a cheese
wedge with an arm extending away.
Icosahedral RNA virusesIcosahedral RNA viruses
Icosahedral RNA virusesIcosahedral RNA viruses
Icosahedral RNA virusesIcosahedral RNA viruses
Assembly of the icosahedral capsid requires intermediate forms and may involve several steps: Poliovirus will be used as an example
Proteolytic cleavage of the polyprotein occurs The basic building block (protomer) of the
capsid is made with Vp0, VP1, and VP3. Five protomers combine to form a pentamer Twelve pentamers combine to form an empty
procapsid RNA enters the procapsid (the arrangement
is not clear, but it is not random)
Assembly of the icosahedral capsid requires intermediate forms and may involve several steps: Poliovirus will be used as an example
Proteolytic cleavage of the polyprotein occurs The basic building block (protomer) of the
capsid is made with Vp0, VP1, and VP3. Five protomers combine to form a pentamer Twelve pentamers combine to form an empty
procapsid RNA enters the procapsid (the arrangement
is not clear, but it is not random)
Icosahedral RNA virus AssemblyIcosahedral RNA virus Assembly
A maturation cleavage converts VP0 into VP2 and VP4
The loops and carboxy termini face the outside of the capsid, while the amino termini and VP4 face the inside of the capsid.
Remember: After attachment of the virus to the host cell, VP4 is released and this exposes a hydrophobic domain on VP1 which interacts with the either the plasma membrane of the host cell or the membrane of an endocytic vesicle following endocytosis, to create a pore through which the viral nucleic acid is released into the cytoplasm.
A maturation cleavage converts VP0 into VP2 and VP4
The loops and carboxy termini face the outside of the capsid, while the amino termini and VP4 face the inside of the capsid.
Remember: After attachment of the virus to the host cell, VP4 is released and this exposes a hydrophobic domain on VP1 which interacts with the either the plasma membrane of the host cell or the membrane of an endocytic vesicle following endocytosis, to create a pore through which the viral nucleic acid is released into the cytoplasm.
Poliovirus AssemblyPoliovirus Assembly
Poliovirus AssemblyPoliovirus Assembly
Icosahedral DNA Virus Assembly
Icosahedral DNA Virus Assembly
SV40 The capsid of SV40 virus has an
unusual arrangement that appears to violate the rules for icosahedral shapes.
Rather than having the classic 12 pentamers and 60 hexamers, all capsomers are grouped into pentamers.
SV40 The capsid of SV40 virus has an
unusual arrangement that appears to violate the rules for icosahedral shapes.
Rather than having the classic 12 pentamers and 60 hexamers, all capsomers are grouped into pentamers.
SV40 AssemblySV40 Assembly
SV40 structureSV40 structure
AssemblyAssembly
There is very little known about the assembly events of other icosahedral DNA animal viruses.
It is clear, however, that the assembly takes place in a very ordered sequence of events.
There is very little known about the assembly events of other icosahedral DNA animal viruses.
It is clear, however, that the assembly takes place in a very ordered sequence of events.
Adenovirus assemblyAdenovirus assembly
AssemblyAssembly How do viruses with segmented genomes ensure
that virions contain a copy of each segment? The answer is simple for some – they don’t. For others, there appear to be specific mechanisms for
packaging their segmented genomes. Each segment may have its own unique pac site.
For influenza virus the ratio of virus particles to actual infectious units is comparable to the ratio predicted for random packaging. However recent evidence suggests that during
budding, viral proteins recognize and interact with specific RNA sequences in each of the eight nucleocapsids.
They then incorporate them, one by one, into bundles that are packaged into virions during budding
How do viruses with segmented genomes ensure that virions contain a copy of each segment? The answer is simple for some – they don’t. For others, there appear to be specific mechanisms for
packaging their segmented genomes. Each segment may have its own unique pac site.
For influenza virus the ratio of virus particles to actual infectious units is comparable to the ratio predicted for random packaging. However recent evidence suggests that during
budding, viral proteins recognize and interact with specific RNA sequences in each of the eight nucleocapsids.
They then incorporate them, one by one, into bundles that are packaged into virions during budding
ReleaseRelease
How do viruses exit their host cells? Naked viruses
If the virus lyses the host cells, it is said to be cytocidal or cytolytic.
For animal viruses lysis is due to the cumulative metabolic damage to the cell caused by the virus.
Disruption of lysosomes may be involved.
How do viruses exit their host cells? Naked viruses
If the virus lyses the host cells, it is said to be cytocidal or cytolytic.
For animal viruses lysis is due to the cumulative metabolic damage to the cell caused by the virus.
Disruption of lysosomes may be involved.
ReleaseRelease
Enveloped viruses The envelopes are derived from host cell membranes
that have been modified by the insertion of viral proteins and glycoproteins
Maturation and release via the process of budding (exocytosis) involves 4 steps
Synthesis and insertion of viral glycoproteins in host cell membranes (RER, Golgi, PM, nuclear membrane)
Assembly of the viral nucleocapsid The nucleocapsid and the modified membrane are
brought together (the C terminal domain of the envelope proteins may interact directly with the nucleocapsid or the interaction may be via the matrix (M) protein)
Exocytosis or budding which may or may not kill the host cell
Enveloped viruses The envelopes are derived from host cell membranes
that have been modified by the insertion of viral proteins and glycoproteins
Maturation and release via the process of budding (exocytosis) involves 4 steps
Synthesis and insertion of viral glycoproteins in host cell membranes (RER, Golgi, PM, nuclear membrane)
Assembly of the viral nucleocapsid The nucleocapsid and the modified membrane are
brought together (the C terminal domain of the envelope proteins may interact directly with the nucleocapsid or the interaction may be via the matrix (M) protein)
Exocytosis or budding which may or may not kill the host cell
BuddingBudding
BuddingBudding
How does a virus target a particular membrane region as the site of budding?
They utilize the transport machinery of the host cells
Some viruses bud from the plasma membrane As they exit the cell they acquire their
membrane.
How does a virus target a particular membrane region as the site of budding?
They utilize the transport machinery of the host cells
Some viruses bud from the plasma membrane As they exit the cell they acquire their
membrane.
Influenza virus budding from the plasma
membrane
Influenza virus budding from the plasma
membrane
BuddingBudding
Some viruses bud from the RER. Some viruses bud from the Golgi. Some viruses bud from the nuclear envelope.
How do viruses that don’t bud from the plasma membrane exit the cell?
Some viruses bud from the RER. Some viruses bud from the Golgi. Some viruses bud from the nuclear envelope.
How do viruses that don’t bud from the plasma membrane exit the cell?
Budding From RER or Golgi
Budding From RER or Golgi
BuddingBudding The plasma membranes of some host cells are
polarized. If the cell is polarized, the virus may bud from
either the apical or the basolateral domain. Viruses that bud apically tend to cause
localized infections. Viruses that bud basolaterally tend to cause
systemic infections. The envelope proteins of these viruses contain
apical or basolateral transport signals that are recognized and utilized by the transport machinery of the host cell.
The site of envelope protein transport determines the site of budding.
The plasma membranes of some host cells are polarized.
If the cell is polarized, the virus may bud from either the apical or the basolateral domain.
Viruses that bud apically tend to cause localized infections.
Viruses that bud basolaterally tend to cause systemic infections.
The envelope proteins of these viruses contain apical or basolateral transport signals that are recognized and utilized by the transport machinery of the host cell.
The site of envelope protein transport determines the site of budding.
Polarized BuddingPolarized Budding
Maturation of virus particles
Maturation of virus particles
For most viruses, formation of the infectious virions requires the cleavage of precursor proteins into functional proteins. The cleavage may occur before assembly.
The HA of influenza virus is cleaved into HA1 and HA2 during transit of the protein to the host cell plasma membrane.
The cleavage may occur after assembly Remember the cleavage of polio virus VP0 into VP2
and VP4?
For most viruses, formation of the infectious virions requires the cleavage of precursor proteins into functional proteins. The cleavage may occur before assembly.
The HA of influenza virus is cleaved into HA1 and HA2 during transit of the protein to the host cell plasma membrane.
The cleavage may occur after assembly Remember the cleavage of polio virus VP0 into VP2
and VP4?
