Terms •Capsid •Envelopes •Nucleocapsid

advertisement
Lecture 3. Physical Structures
Terms
•Capsid
•Envelopes
•Nucleocapsid
•Helical
•Icosahedral
Great website: http://viperdb.scripps.edu/
1
Size, mass, dimensions of viruses
Particle- An aggregate of many molecules often of
different nature.
•Weights range from 3-800 X 106 Daltons (Da).
•Linear dimensions generally given in nm (10-9 meters).
Typical viruses range from ≈ 25 - >100 nm diameter.
•Comparison: Bacterial cells: ≈1500 nm dia; Eukaryotic
epithelial cells about ≈20,000 nM dia.
•Assuming viruses and bacteria are nearly spherical
(not always the case), a bacteria has a volume about
30,000 times greater than a virus while a epithelial cell
is about 60 million times larger.
• could stuff about 3 x 1014 virus particles in one
drop of water.
2
Size, mass, & dimensions
• S-value- A value derived from the
sedimentation rate of particles and molecules
in the ultracentrifuge.
• Numbers are reproducible under specific
conditions and reflect the volume and shape
of a particular substance but are not directly
proportional to mass.
• The basic unit is the Svedberg (S) which is
10-13 sec.
• This value can be used to estimate molecular
weights in conjunction with other values.
3
Structural Principles and
terms
• Helical-Rod or threadlike appearance
• Isometric-spherical appearance
• Irregular-without clear symmetry
4
Virus makeup
• Protein subunit: individual folded protein molecules.
• Structural subunit (synonyms; protomer, asymmetric unit): Unit
from which capsid of nucleocapsid are built; may comprise one
protein subunit or multiple different subunits.
• Morphological unit (syn: capsomere): surface structure (knobs,
projections, clusters, etc.) seen by electron microscopy. This
term is generally restricted to descriptions of viruses from
electron micrographs.
• Capsid (syn: coat): regular, shell-like structure composed of
aggregated protein subunits which surrounds the viral nucleic
acid.
• Nucleocapsid (syn: core): viral nucleic acid enclosed by a capsid
protein coat.
• Envelope (syn: viral membrane): lipid bylayer containing viral
glycoproteins. The phospholipids in the bylayer are derived
from the cell that the virus arose from. Not all viruses have
envelopes some consist of only the nucleocapsid.
• Virion: physical virus particle. Nucleocapsid alone for some
viruses (picornaviruses) or including outer envelope structure
for others (retroviruses).
5
The Viral Capsid
• Capsid- Protein coat that encapsidates the virus.
• Nucleocapsid-Capsid with genome inside (plus anything
else that may be inside like enzymes and other viral
proteins for some viruses).
Capsid functions
1. Protect genome from outside environment (May include
damaging UV-light, shearing forces, nucleases either
leaked or secreted by cells).
2. Virus-attachment protein- interacts with cellular
receptor to initiate infection. Since viruses are made of
many different repeated subunits there is redundancy;
Many receptor sites so damage to a few doesn’t prevent
infection.
3. Delivery of genome in infectious form. May simply
“dump” genome into cytoplasm (most +ssRNA viruses) or
serve as the core for replication (retroviruses and 6
rotaviruses).
How do particles form?
• Information is encoded in the components themselves
(nucleic acid + proteins).
• Some proteins can form capsid shells in the absence
of the genome; others form around the genome.
• Fraenkel-Conrat and Williams (1955): TMV
nucleocapsids form spontaneously from individual
protein subunits (coat protein) and the genome.
•  Particles represent a free energy minimum state
which leads to stability.
• Assembly is driven by hydrophobic and hydrophilic
(rarely covalent) interactions including
– Protein-protien,
– Protein-nucleic acid
– Protein-lipid
• Important: since particles must disassemble at some
point during infections, covalent bonds would make 7
this more difficult.
• Why not make the capsid from a single large
protein rather than assemble it from many
proteins?
• Not enough genomic information:
• MW of 1 codon (3 nucleotides) is about 1000 Da.
1 amino acid is about 110 Da, a genome can only
produce proteins that are 10% of its molecular
weight.
• A picornavirus is about 10,000 bases----can
produce a protein about 500,000 Dal.
• Outer shell of picornaviruses is made up of 60
copies each of 4 different proteins, approx
MW=2 million Da.
The genome does not have enough information to
8
encode a single protein that could encapsidate it.
Virus Shapes
• Most viruses have evolved to form one of 2 different
shapes;
• Helical and Icosohedral.
• Some irregular viruses do exist and many of these
have underlying helical or icosohedral symmetry.
• Note-viruses form regular shapes but use irregular
proteins to do so.
• This creates a problem that must be solved for
assembly to occur.
• For example, it would be easy to imagine how a virus
might form an icosahedron if perfectly triangular
proteins were used. But the proteins are irregular
shaped and still must form a sealed (to protect
genome) icosahedron.
9
Helical nucleocapsids
Topology follows the biophysical geometry of the nucleic acid
genome.
Enveloped Helical
nucleocapsid
Schematic representation
of Tobacco Mosaic Virus.
EM of Influenza C
filamentous particle
10
Helical viruses
• Many biological components have adopted a helical
structure (DNA, a helix of proteins).
• Energetically favorable and can allow flexibility (bend but
don’t break (shear)).
• The simplest way to arrange irregular identical proteins
would be around a central axis to form a disk. Disks
could then be stacked with the genome in the middle to
form a cylinder.
• Helical viruses form a closely related spring like helix
instead. The best studied TMV but many animal viruses
and phage use this general arrangement.
– Note-all animal viruses that are helical are enveloped, unlike many
of the phage and plant viruses.
• Most helixes are formed by a single major protein
arranged with a constant relationship to each other
(amplitude and pitch).
• They can be described by their Pitch (P, in nm):
• P= m x p, m-# of protein subunits per helical turn, p-axial
11
rise per subunit
VSV, a prototypical helical animal virus
• VSV (Vesicular stomatitis virus) coat protein: a “typical”
helical coat protein.
• Small (50 aa derived from a 73 aa precursor),
• alpha helical with 3 distinct domains
• Domain characteristics are consistent with their function,
• + charge interacts with nucleic acid,
• hydrophobic with proteins on either side,
• negative charge with polar environment.
• Subunits are tilted 20o relative to the long axis of the
particle. P= 6.75 nm, m=4.5, and p=1.5.
• VSV Genome: 11,000 nt -ssRNA interacts with the
nucleocapsid protein (N) to form a helical structure with
P=5 nm.
• The particle is about 180 nm long and 80 nm wide.
Fig 4.4
12
13
14
Icosahedral structures
Basic soccer ball and variations.
Topology follows the constraints of Euclidean solid geometry.
5
3
The icosahedron:
12 vertices, 20 triangular faces,
30 edges.
2
5- , 3-, and 2-fold rotational axes.
5-fold rotational axes pass
through the vertices.
3-fold pass through centers of
the triangular faces.
2-fold pass through the edges.
15
Triangulation number (T)
DO NOT MEMORIZE THIS EQUATION!!!!
•T=f2 x P where f=# of subdivisions on each side of a triangular face, P=h2
+ hk + k2 where h and k are any nonnegative integer.
•Only T’s that may be derived from the above equation are possible.
•60 = minimal number of irregular subunits required
•Beyond 60 subunits equivalence is not possible.
T=3
T=4
T=7l
T=13l
16
17
18
Picornaviridae, a prototype T=3 virus
• Fig. 4.9A illustrates quasi-equivalence with pentamer
at each vertex and hexamers in other regions;
• Triangulation # = 3.
• Note that VP-4 is not on the surface of the structure
but lies under the face.
19
Picornaviridae, a prototype T=3 virus
• Fig. 4.9B. The protein subunits that form each protomer all
assume a similar (not identical) shape .
• In fact all T=3 RNA viruses have proteins that form “8 strand
antiparallel b barrels”.
• The structures form from the polypeptide by first forming a
“jelly-roll barrel” that then goes on to form the wedge-shaped
barrel when the capsid is being formed.
20
21
22
Complex structures
• Many, viruses are so large that they have evolved
complex, more “cell like” structures.
• e.g. Poxviridae, paramyxoviridae, Coronaviridae)
Vaccinia virus, from Fig. 13.16
23
How do capsids interact with the
genomic RNA rather than other cellular
RNA?
• In many cases viruses produce huge amounts
of viral genome and inhibit cell mRNA
synthesis (either directly or indirectly). This
helps but does not solve the problem.
• Viral genomes have packaging signals (“psi” or
Y) that form structures recognized by one of
the capsid proteins.
• Keep in mind that viruses never do anything
correct 100% of the time (defective
particles).
24
Where do virus capsids form in the cell?
• Nonenveloped viruses
– Generally form in the cytoplasm (some in the nucleus)
and mature before being released when the cell lyses.
• Enveloped Viruses-
– Generally form the capsid as they are budding from a
membrane.
– Envelope proteins are inserted into the host cell
membrane prior to budding.
– Viral matrix and capsid proteins interact with the
membrane and membrane proteins at regions with a
lot of envelope proteins.
– The genome interacts with the nucleocapsid protein
and budding is initiated.
– Often the virion matures after release.
25
From: http://home.ncifcrf.gov/hivdrp/Freed_figure.html
26
Download