1/16/13 VIRUS STRUCTURE

Sergei Nekhai, Ph.D.

Objectives:

•Functional organization of viral particles

• Viral Symmetry

• Viral Capsids

Structure of Viruses

• Size range –

– most

Electron Microscopy

•Staining with electron-contrast (phosphotungstic acid or uranyl

acetate) material

•Evaluation of stool specimens from patients with gastroenteritis

(rotaviruses, astroviruses, adenoviruses, noroviruses)

• Direct detection of viral particles when viral culture conditions

or reagents are not available (early detection of SARS)

•Direct examination of specimens for herpesvirus, poxvirus or

Ebola

•Examination of fixed tissues obtained from biopsy or autopsy if

histology is performed as well

•Examination of infected tissue culture

Electron Microscopy

Scanning Electron Microscopy

Cryo-Electron Microscopy

•Any waveform can be presented as a sum of simple

sinusoids of different frequencies

•The Fourer Transform decomposes any waveform

into sinusoids

Combine

FT of

Single

images

Inverse FT

and 3D

reconstruction

Dryden, et al, 1993. J. Cell Biol. 122:1023-1041.

Mammalian Reovirus

X-Ray Chrystallography

Structure of Viruses

(Baker et al.)

Characteristic size scale is 30-100 nm.

Structures are known at “atomic resolution” - see Viper website (http://viperdb.scripps.edu/

Highly symmetric - think hard about what this implies about assembly!

http://viperdb.scripps.edu/

Viruses

Figure 13.1

Organization of Viral Particles

•Contains RNA or DNA

•Form a protective package

•Transmit genetic material

•Entry, multiply and exit

the host

•Redirect cellular

machinery

E. coli Streptococcus

Yeast Cell

Terminology

• Virion: physical virus particle. Nucleocapsid alone for some viruses (picornaviruses) or including outer envelope structure for others (retroviruses).

• 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

Viruses - Structure

• contain DNA or RNA

• contain a protein coat (capsid)

• Some are enclosed by an envelope

• Some viruses have spikes

General Structure of Viruses

• Capsids

– All viruses have capsids - protein coats that enclose

and protect their nucleic acid.

– Each capsid is constructed from identical subunits

called capsomers made of protein.

– The capsid together with the nucleic acid are

nucleoscapsid.

The Viral Capsid• Capsid- Protein coat that encapsidates the viral genome.

• 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 atmosphere (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.

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 rotaviruses).

Human Viruses

"Group" Family Genome Genome size (kb) Capsid Envelope

dsDNA

Poxviridae dsDNA, linear 130 to 375 Ovoid Yes

Herpesviridae dsDNA, linear 125 to 240 Icosahedral Yes

Adenoviridae dsDNA, linear 26 to 45 Icosahedral No

Polyomaviridae dsDNA, circular 5 Icosahedral No

Papillomaviridae dsDNA, circular 7 to 8 Icosahedral No

ssDNA

Anellovirus ssDNA circular 3 to 4 Isometric No

Parvoviradae ssDNA, linear, (- or +/-) 5 Icosahedral No

Retro

Hepadnaviridae dsDNA (partial), circular 3 to 4 Icosahedral Yes

Retroviridae ssRNA (+), diploid 7 to 13 Spherical, rod or cone shaped Yes

dsRNA

Reoviridae dsRNA, segmented 19 to 32 Icosahedral No

ssRNA (-)

Rhabdoviridae ssRNA (-) 11 to 15 Helical Yes

Filoviridae ssRNA (-) 19 Helical Yes

Paramyxoviridae ssRNA (-) 10 to 15 Helical Yes

Orthomyxoviridae ssRNA (-), segmented 10 to 13.6 Helical Yes

Bunyaviridae ssRNA (-, ambi), segmented 11 to 19 Helical Yes

Arenaviridae ssRNA (-, ambi), segmented 11 Circular, nucleosomal Yes

Deltavirus ssRNA (-) circular 2 Spherical Yes

ssRNA (+)

Picornaviridae ssRNA (+) 7 to 9 Icosahedral No

Calciviridae ssRNA (+) 7 to 8 Icosahedral No

Hepevirus ssRNA (+) 7 Icosahedral No

Astroviridae ssRNA (+) 6 to 7 Isometric No

Coronaviridae ssRNA (+) 28 to 31 Helical Yes

Flaviviridae ssRNA (+) 10 to 12 Spherical Yes

Togaviridae ssRNA (+) 11 to 12 Icosahedral Yes

Principles of Viral Architecture

•Viral capsid are made of repated protein subunits

•Capsids are self assembled

•Fraenkel-Conrat and Williams (1955): self-assembly of TMV

•Proteins and nucleic acids are held together with non-

covalent bonds

•Protein-protein, protein-nucleic acid, protein-lipid

•Helical or icosahedral symmetry

Viral Capsids

• If 1 protein for 1 capsid:

– Need > 18,000 amino acids.

– Need > 54,000 nucleotides.

– Small viruses hold max. of 5,000 nucleotides.

• Must use many copies of 1 (or a few) protein(s).

• High symmetry

– Minimizes # different subunit interactions involved with assembly.

– Simpler protein.

– Self assembly:

• Self-contained assembly "instructions".

Basic Nucleocapsid Structures:

• HELICAL: Rod shaped, varying widths and specific

architectures; no theoretical limit to the amount of nucleic

acid that can be packaged

• CUBIC (Icosahedral): Spherical, amount of nucleic acid

that can be packaged is limited by the number of

capsomers and the size of the viral particle

• Irregular: Without clear symmetry

Capsid and Envelope

Non-enveloped

Icosahedral Enveloped

Helical

Capsid:•Protect viral nucleic acid

•Interact with the nucleic acid for packaging

•Interact with vector for specific transmission

•Interact with host receptors for entry to cell and to release of nucleic acid

Envelope:•Made from host cell membrane (plasma, ER or Golgi)

•Fuse for Entry

Helical viruses• Organized around a single axis (the “helix axis”)

• Probably evolved along with other helical structures like DNA, a-helix, etc.

• Allow flexibility (bending)

• 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= u x p, u-# of protein subunits per helical turn, p-axial rise per subunit

Helical symmetry

• Tobacco mosaic virus is typical,

well-studied example

• Each particle contains only a single

molecule of RNA (6395 nucleotide

residues) and 2130 copies of the

coat protein subunit (158 amino

acid residues; 17.3 kilodaltons)

– u=16.33 subunits/turn

– p=1.4 Å

– P= 23 Å

• TMV protein subunits + nucleic

acid will self-assemble in vitro in an

energy-independent fashion

• Self-assembly also occurs in the

absence of RNA

TMV rod is 18 nanometers (nm) X 300 nm

Influenza virus

Ebola Virus• Filamentous Filovirus with single-stranded (-) RNA genome

• The capsid has a helical morphology and is encased inside a membrane

envelope.

• VP30- matrix protein; L protein – RNA polymerase

Vesicular Stomatitis Virus• VSV coat protein (50 aa): alpha helical with 3 distinct domains:

+ 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.

• VSV Genome: 11,000 nt -ssRNA interacts with the nucleocapsid

protein (N) to form a helical structure with P=5 nm. .

•

ICOSAHEDRAL VIRUSES

•1956, Watson and Crick – only cubic symmetry

leads to isometric particle

•Only three cubic symmetry exist:

•tetrahedral (2:3) – 12 identical

subunits

•octahedral (4:3:2) – 24 identical

subunits

•icosahedral (5:3:2) – 60 identical

subunits

•For viruses of 150-200 Å - ~ 60 of

20 kDa protein subunits

•However, for viruses > 250 Å (turnip

yellow mosaic), it was more than 60

subunits

Parvovirus Structure

Picornavirus Structure

QUASI-EQUIVALENCE

1962, Caspar and Klug – found a principal of building

icosahedral structures from similar blocks

• Shell is built from the same blocks

•Bonds are deformed in a slightly different ways

•Assumed a possibility of 5 degrees deformation

•Shell can contain 60n subunits

A Fuller geodesic dome

That inspired Caspar

and Klug

Triangulation number (T) Enumerated by Caspar

and Klug

• 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

CLASSES OF ICOSAHEDRAL DELTAHEDRA

Tabulation of the Triangulation Number T

Class

P = 1 1 4 9 16 25 . . . .

P = 3 3 12 27 . . . .

Skew Classes 7 13 19 21 . . . .

T = Pf2, where P = h2 + hk + k2, h and k any pair of integers with no common

factor, and f= 1 , 2 , 3 , 4 , . . . .

Number of structure units S=60 T

Number morphological units M = 10 T + 2= 10(T-1) hexamers + 12 pentamers

(a) P=1, T=1. (b) P = 1, T= 4. (c) and (d), P = 3 (T=3 and 12, respectively). (e), (f),

(g) and (h), first members of the skew classes P = 7, 13, 19, and 21, respectively.

CLASSES OF ICOSAHEDRAL DELTAHEDRA

Different Arrangements of Icosahedral Symmetry

Zlotnick A. PNAS 2004;101:15549-15550

©2004 by National Academy of Sciences

Jellyroll: Many, but not all Viral

proteins

Capsid proteins

b-barrel.

• Rhombohedral wedges:

– Fit into icosahedron.

• Jellyroll topology

• Conserved in many small viruses

– T = 1, 3, …

• 60, 180, 240 proteins…

– RNA or DNA viruses.

• Essentially no sequence homology.

Picornaviridae, a prototype T=3 virus• 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.

Picornaviridae, a prototype T=3 virus• 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.

• Each particle contains

only a single molecule

of RNA (4800 nt) and

180 copies of the coat

protein subunit (387 aa;

41 kd)

• Viruses similar to TBSV

will self-assemble in

vitro from protein

subunits + nucleic acid

in an energy-

independent fashion

TBSV icosahedron is 35.4 nm in diameter

Protein Subunits Capsomeres

T= 3 LatticeC

N

Tomato Bushy Stunt Virus

Assembly of Turnip Crinkle Virus

Scaffold-guided Assembly of Bacteriophage HK97

T=7

420 subunits