Transcript Assembly, Maturation, and Release - Cal State LA

Assembly, Maturation, and
Release
(Getting it all together and
leaving!)
Assembly
 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.
Assembly
 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.
Assembly
 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.
TMV
TMV 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.
TMV assembly
Simple assembly model
TMV 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.
TMV assembly
TMV 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.
TMV assembly
Assembly
 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 paclike regions to assure that only the correct RNA is
assembled in the nucleocapsid.
Assembly
 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 viruses
Icosahedral 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.
Icosahedral RNA viruses
Icosahedral RNA viruses
Icosahedral 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)
Icosahedral 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.
Poliovirus Assembly
Poliovirus 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 Assembly
SV40 structure
Assembly
 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 assembly
Assembly
 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
Release
 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.
Release
 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
Budding
Budding
 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
Budding
 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
 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 Budding
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?