Viral Capsids

Question 1

Viruses can not live without a host cell

Answer 1

• Use cell (energy, proteins, compartments, molecular machines, etc.) for replication i.e. transcription and translation of viral genes and for progeny virus particle assembly
• All viruses, with transmissibility via extracellular routes, contain a capsid (virus encoded), many also a lipid bilayer membrane (envelope of cellular origin, e.g. ER, Golgi, cell wall)
• Have evolved different strategy for entry into and exit from the host cell:
• Enveloped involves membrane fusion
• Non-enveloped display more complex strategies and some have very elaborated structures (Bacteriophage T4)
  p.s. capsid-less RNA viruses include the families Narnaviridae, Hypoviridae, and Endornaviridae

Question 2

Viral Capsids

Answer 2

• Protect against physical, chemical, and enzymatic damage
• Economy, a few protein subunits are needed/repeated: - small particle size and thus small genome size since a nucleic acid can never code for a single capsid protein molecule that is big enough to enclose and protect the whole genome
• The simplest viruses have just one type of protomer
• Porcine circovirus (PCV) is about 17 nm, Mimivirus about 400 nm (1.18 MB Genome; Chlamydia trachomatis 1.05 MB)
• Rod-shaped or roughly spherical shell (icosa- or dodecahedron, i.e. 20 triangles or 12 pentagons)

Question 3

More on enveloped virus particles

Answer 3

• helical or icosahedral struc- ture underlying the envelope may be formed before or during /after (ripening) the virus leaves the host cell
• many viruses assemble in the cell surface membrane, but others use cytoplasmic mem- branes such as the Golgi, or the nuclear membrane (e.g. HSV) -> virus is transported in vacuole

Question 4

Enveloped helical virus particles

Answer 4

• Rhabdovirus (e.g., Rabies)
• Orthomyxoviridae (Influenza)
• Paramyxoviridae (Mumps, Measles)
  -> (-)ssRNA viruses

Question 5

Helical viruses
-> Helical Symmetry

Answer 5

Tobacco mosaic virus (TMV) is the “prototype” helical virus, made up of a spiral or helix of about 2,200 protein subunits (jelly roll), set like shallow steps in a spiral staircase, making 130 turns around the central core of the particle, which is about 300 nm long with a pitch (P) of 2.3 nm. Each turn of the helix is made of 16.3 protein subunits which enclose a continuous strand of RNA - the genome. The diameter of the particle is 18 nm, and the hollow core down the centre of the particle is 2 nm in diameter.
- the simplest way to arrange multiple, identical protein subunits is to use rotational symmetry -> helix -> defined by amplitude and pitch -> number of subunits per turn, µ, and axial rise per subunit, p
-> pitch or P of the helix: P = µ x p
- for TMV, µ = 16.3 -> 16.3 coat protein molecules per helical turn; p = 0.14 nm -> pitch (P) of the TMV helix is 16.3 x 0.14 = 2.28 nm (CP -> 316 residues, 35 kDA)
Many bacteriophages have helical capsids
- phage M13 ! length 900 nm, diameter 9 nm
- particles contain 5 proteins
μ = 4.5 (units/turn)
p = 1.5 nm (axial rise) P = 4.5 x 1.5 = 6.75 nm
- g8p is the major coat protein -> 2700 - 3000 copies -> 50 residues, almost entirely helical -> molecule is like a short rod

Question 6

Symmetrical Polyhedra

Answer 6

The 3D regular convex polyhedra organised according to their symmetry group.
N0 is the number of vertices,
N1 is the number of edges and
N2 is the number of faces constituting the solid.

Question 7

Spherical viruses

Answer 7

1) Specificity = subunits must recognize each other with precision, because virus particles assemble spontaneously from individual components
2) genetic economy: many copies from a few kinds of subunits -> Symmetry!

Icosa- and dodecahedron allow a maximum number of identical objects to form a closed symmetrical shell -> identical symmetries but different shapes

• Spherical viruses to date display icosahedral symmetry
• Adenovirus Dodecahedron in replication cycle
• genome can form a dodecahedral cage
• immature/empty shells can display dodecahedral symmetry
• Icosahedron is built from 20 identical equilateral triangles
• Triangular tiles are arranged side by side so that they enclose the volume inside the icosahedron

The icosahedron has:
- 12 vertices -> each with a 5-fold rotation axis
- 20 faces (tiles) -> each with a 3-fold rotation axis through the middle (asymmetric unit)
- 30 edges -> each with a 2-fold rotation axis through the middle
- an icosahedron can be divided into a number of smaller identical pieces called symmetry-related units -> these are the 20 tiles -> 3-fold (non-crystallographic) symmetry
- but: protein chains are considered asymmetric objects -> a symmetry axis cannot pass through them

• the tiles have 3-fold symmetry -> 3 identical objects are needed to form one tile (economy rules) -> total number of objects in an icosahedron is 20 x 3 = 60 -> 60 asymmetric units
• same “result” for 12 corners with 5-fold symmetry, or 30 edges with 2-fold symmetry (12x5=30x2=60)
• the protein shell of a spherical virus with icosahedral symmetry must have a minimum of 60 identical units
• is the volume inside a shell of only 60 proteins large enough to accommodate the viral genome?
  -> very few self-sufficient viruses have only 60 protein chains in their shells

Question 8

Spherical viruses
-> Satellite viruses

Answer 8

• but satellite viruses with only 60 proteins do occur
• satellite viruses do not encode all of the functions required for their replication -> they are not self-sufficient
• satellite tobacco necrosis virus -> diameter of only 180 Å -> protein shell of only 60 subunits -> RNA genome has only 1121 nucleotides
  -> cannot replicate on its own inside a tobacco cell -> needs a helper virus ! tobacco necrosis virus -> satellite viruses are obligate parasites of viruses that parasitize cells

Question 9

Spherical viruses
-> Self-sufficient viruses

Answer 9

• self-sufficient viruses have longer genomes ! code for enzymes essential for replication of the viral nucleic acid, in addition to the structural proteins -> larger volume inside the capsid required
• asymmetric unit must contain more than one subunit
• these subunits can be identical or different -> genetic economy would favour identical subunits…
  -> only certain multiples (1,3,4,7…) of 60 subunits are likely to occur
  -> triangulation number, T

T = h^2 + hk + k^2

Question 10

Spherical viruses
-> T = 3 viruses

Answer 10

• T = 3 virus -> 180 subunits -> A subunits interact around the 5-fold axes, and B and C interact around the 3-fold axes -> 3 subunits each of B and C arranged in a pseudosymmetric way -> 3-fold becomes a pseudo 6-fold

Question 11

Spherical viruses
-> T = 4 viruses

Answer 11

• 240 subunits in 4 different environments
  -> A, B, C, D in the asymmetric unit -> A subunits interact around the 5- fold, D subunits around the 3-fold
  -> B, C subunits are arranged such that two copies of each interact around the 2-fold, in addition to two D subunits
  -> the 2-folds form pseudo 6-fold axes
  -> the A, B, C subunits interact around pseudo 3-fold axes clustered around the 5-folds
  -> 60 such pseudo 3-fold axes

T = 4: HBV, CHIKV, Rubella

Question 12

Tomato bushy stunt virus (T = 3)

Answer 12

• 180 chemically identical polypeptide chains, each of 386 residues
• 3 domains in each protomer: R (internal, disordered), S (viral shell), P (protruding out from the surface); connecting regions: a and h
• These subunits pack into the virus particle in one of three conforma- tions -> A (red), B (blue), C (green)
• C molecule, in particular, is different
  -> hinge between S and P different
  -> subunits adapt to the 3 different environments
  -> connecting region a is ordered in C only
• P domains interact pair wise across 2-fold axes -> protrusions
• 30 twofold axes relating P domains of subunit C (green), plus 60 pseudo twofold axes relating P domains of subunits A (red) to P domains of subunit B (blue)
  -> the 180 P domains form 90 protrusions
• ordered connecting arms a of subunit C interdigitate (dodecahedron?)
  -> internal framework along the inner face of the S domain
  -> loop around icosahedral 3-fold axes
• this framework determines the size of the particle
  -> diameter of TBSV is 330 Å (compared to 180 Å for STNV)
  -> an RNA molecule about 4 times larger can be accommodated

Question 13

Picornaviruses

Answer 13

• shell of all picornaviruses is built from 60 copies each of 4 polypep- tides, VP1 to VP4
  -> translated from viral RNA as a single polypeptide
  -> post-translationally processed by stepwise proteolysis involving virally encoded protease-polymerase unit (3CD)
  -> 1st step: VP0, VP1, VP3 -> assembly of shell begins -> VP0 precursor is cleaved into VP2 and VP4
• molecular masses: VP1, VP2, VP3: 30 kD; VP4: 7 kD -> completely buried inside the particle (T = 3 capsid despite 4 protomers!)
• VP1, VP2, VP3 have no significant sequence identity
• yet, the 180 subunits are arranged in a very similar way to the 180 subunits (built from one type of polypeptide) of the plant T=3 viruses
• this is achieved not by conformational differences as in the plant viruses, but by having 3 chemically different polypeptide chains with local structural differences
• asymmetric unit: one copy each of VP1, VP2, VP3, VP4 (buried inside)
  virus shell built from 12 pentamers -> “molecular mountain“

Question 14

3D structures of coat proteins of plant and picornaviruses

Answer 14

• very common: “jelly roll“
• b-strands are arranged into two separate sheets: strands 1, 8, 3, 6 and strands 2, 7, 4, 5
• space between the sheets is filled by hydrophobic side chains
• example: S domain of tomato bushy stunt virus

Question 15

The Greek key motif

Answer 15

” A “Greek key“ is formed when one of the connections of four antiparallel b-strands is not a hairpin connection, but strand n is connected to strand n+3 (a) or n-3 (b) instead of n+1 or n-1 in an eight-stranded b-sheet or barrel (only (a) has been observed to date).

Question 16

The jelly roll

Answer 16

• resembles a piece of string wrapped around a barrel
• in the jelly roll motif, there are four connections across the end of the barrel, the polypeptide chain enters and leaves the barrel at the same end
• the polypeptide chain has 8 straight sections (b-strands), interrupted by loop regions

Question 17

Jelly roll structures of viral coat proteins

Answer 17

• fairly large number of residues at the chain termini outside the barrel (bottom of barrel; white)
  -> vary considerably in size and conformation between different coat proteins
• loops at this end of the barrel are quite long -> considerable variation
• in contrast, the 4 loop regions at the top of the barrel are short
  -> all coat proteins are formed like a wedge

Canyon binder drugs (VP1) prevent uncaring of the viral capsid

Question 18

The bacteriophage MS2 has a coat protein with a different fold

Answer 18

• ssRNA phage, infects E. coli
• T=3 virus, diameter about 250 Å
• genome has 4000 nt, encodes only 4 proteins
• one of them is coat protein -> 129 residues
• another protein is present in a single copy per virus particle -> essen- tial for host attachment and penetration of the ssRNA
• coat protein folds into a 5-stranded up-and-down b-sheet + N-terminal hairpin + two C-terminal helices
• C-terminal a-helices responsible for dimer formation
• b-sheets align at their edges -> 10-stranded b-sheet
  -> structure is quite different from jelly roll found in most spherical viruses so far
• MS2 binds to a sequence of 19 nt in the viral genome -> contains the start codon for the phage replicase gene -> recognition controls two processes: - translation of the replicase, - packaging of the RNA genome into the virus shell -> encapsidation of the viral genome is initiated when MS2 dimer binds to this sequence
• 2 of the 4 nt in the loop and the bulged-out adenine are recognized by the MS2 dimer
  -> recognition not only requires correct nucleotide sequence, but also correct stem-loop structure and formation of the MS2 dimer

Question 19

The core protein of alpha virus has a chymotrypsin-like fold

Answer 19

• Alphavirus family: Sindbis virus, Semliki Forest virus, Chikungunya virus -> mosquito-borne, enveloped RNA viruses -> cause encephalitis, fever, arthritis
• subunit of core of Sindbis virus -> differs from jelly roll and MS2
• core protein cleaves itself from large precursor -> has proteolytic Activity!
• core has N-terminal domain that binds RNA, and C-terminal domain that resembles chymo- trypsin -> catalytic triad Ser-His-Asp
• C-terminus of mature coat protein in active site -> deactivation after one cleavage reaction!
• Many viruses also have a lipid bilayer membrane
  -> enclosing the capsid
  -> different strategy for entry into and exit from the host cell
• Enveloped viruses acquire the lipid bilayer when they bud out through the cellular membrane
• Entry by membrane fusion
• Non-enveloped viruses have evolved more complex strategies, and some have very elaborated structures
• Example: Bacteriophage T4 -> must penetrate the bacterial cell wall at specific sites -> inject its DNA into the cell -> resembles a hypodermic syringe

Question 20

Myoviridae

Answer 20

-> bacterio-phages
- Head: icosahedral shell, T = 7 -> attached by a collar to a contractile, helical tail
- plate below tail -> attachment to bacterial host and penetration of cell wall -> lysozymes asso- ciated with plate!
- thin protein fibers attached to plate -> bind to receptors