1/31/11 VIRUS STRUCTURES i N kh i Ph DSergei Nekhai, Ph.D.

Objectives:Objectives:

•Functional organization of viral particles

• Viral Symmetry

• Viral Capsids

Structure of Viruses• Size range –

0 2 i l i– most <0.2 μm; requires electron microscope

Structure of VirusesStructure of Viruses

Characteristic size scale is 30-100 nm.

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

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

(Baker et al.)

Viruses

Figure 13.1

Organization of Viral Particles

•Contains RNA or DNAE coliStreptococcus

•Form a protective package

T it ti t i l

E. coli Streptococcus

•Transmit genetic material

•Entry, multiply and exit Yeast Cell

the host•Redirect cellular machinerymachinery

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 nucleocapsidconsist of only the nucleocapsid

Viruses - Structure

• contain DNA or RNARNA

• contain a protein• contain a protein coat (capsid)

• Some are enclosed b lby an envelope

• Some viruses have spikes

General Structure of Viruses• Capsids

All i h id i h l– All viruses have capsids - protein coats that enclose and protect their nucleic acid.E h id i t t d f id ti l b it– Each capsid is constructed from identical subunits called capsomers made of protein.The capsid together with the nucleic acid are– The capsid together with the nucleic acid are nucleoscapsid.

The Viral Capsidp• 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 e se a ay be s de e e y es a d o e a p o e sfor some viruses).

Capsid functionsCapsid functions1. Protect genome from atmosphere (May include damaging

UV-light, shearing forces, nucleases either leaked or secreted by cells).secreted by cells).

2. Virus-attachment protein- interacts with cellular receptor to initiate infection.

3 Delivery of genome in infectious form May simply “dump”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

G G G ( ) C"Group" Family Genome Genome size (kb) Capsid EnvelopedsDNA

Poxviridae dsDNA, linear 130 to 375 Ovoid YesHerpesviridae dsDNA, linear 125 to 240 Icosahedral YesAdenoviridae dsDNA, linear 26 to 45 Icosahedral NoPolyomaviridae dsDNA circular 5 Icosahedral NoPolyomaviridae dsDNA, circular 5 Icosahedral NoPapillomaviridae dsDNA, circular 7 to 8 Icosahedral No

ssDNAAnellovirus ssDNA circular 3 to 4 Isometric NoParvoviradae ssDNA, linear, (- or +/-) 5 Icosahedral No

RetroHepadnaviridae dsDNA (partial), circular 3 to 4 Icosahedral YesRetroviridae ssRNA (+), diploid 7 to 13 Spherical, rod or cone shaped Yes

dsRNAReoviridae dsRNA, segmented 19 to 32 Icosahedral No

ssRNA (-)Rhabdoviridae ssRNA (-) 11 to 15 Helical YesRhabdoviridae ssRNA (-) 11 to 15 Helical YesFiloviridae ssRNA (-) 19 Helical YesParamyxoviridae ssRNA (-) 10 to 15 Helical YesOrthomyxoviridae ssRNA (-), segmented 10 to 13.6 Helical YesBunyaviridae ssRNA (-, ambi), segmented 11 to 19 Helical YesArenaviridae ssRNA (-, ambi), segmented 11 Circular, nucleosomal YesDeltavirus ssRNA (-) circular 2 Spherical Yes

ssRNA (+)Picornaviridae ssRNA (+) 7 to 9 Icosahedral NoCalciviridae ssRNA (+) 7 to 8 Icosahedral NoHepevirus ssRNA (+) 7 Icosahedral NoAstroviridae ssRNA (+) 6 to 7 Isometric NoAstroviridae ssRNA (+) 6 to 7 Isometric NoCoronaviridae ssRNA (+) 28 to 31 Helical YesFlaviviridae ssRNA (+) 10 to 12 Spherical YesTogaviridae ssRNA (+) 11 to 12 Icosahedral Yes

Principles of Viral Architecture•Viral capsid are made of repated protein subunits

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

•Proteins and nucleic acids are held together with non-covalent bondscovalent 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 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

i i i diff b i i i i l d– Minimizes # different subunit interactions involved with assembly.

– Simpler protein.p p– Self assembly:

• Self-contained assembly "instructions".

Basic Nucleocapsid Structures:p

• HELICAL: Rod shaped varying widths and specificHELICAL: 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• 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 EnvelopeNon-enveloped

Helical

Capsid:•Protect viral nucleic acid•Interact with the nucleic acid for Helicalpackaging•Interact with vector for specific transmission •Interact with host receptors for entry to cell and to release of nucleic acid

Icosahedral Enveloped Envelope:•Made from host cell

b ( lmembrane (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, α-helix, etc.• Allow flexibility (bending) y ( g)• 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

ith t t l ti hi t h th ( lit d dwith a constant relationship to each other (amplitude and pitch).

• They can be described by their Pitch (P, in nm):P # f t i b it h li l t i l i• 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

id id 17 3 kil d lt )acid residues; 17.3 kilodaltons)– u=16.33 subunits/turn– p=1.4 – P= 23

• TMV protein subunits + nucleic acid will self-assemble in vitro in an

i d d t f hienergy-independent fashion• Self-assembly also occurs in the

absence of RNA

TMV rod is 18 nanometers (nm) X 300 nm

Influenza virusInfluenza virus

Ebola Virus• Filamentous Filovirus with single-stranded (-) RNA genome• The capsid has a helical morphology and is encased inside a membrane

envelopeenvelope. • VP30- matrix protein; L protein – RNA polymerase

Vesicular Stomatitis VirusVSV t t i (50 ) l h h li l ith 3 di ti t d i• 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. .•

Human Viruses

G G G ( ) C"Group" Family Genome Genome size (kb) Capsid EnvelopedsDNA

Poxviridae dsDNA, linear 130 to 375 Ovoid YesHerpesviridae dsDNA, linear 125 to 240 Icosahedral YesAdenoviridae dsDNA, linear 26 to 45 Icosahedral NoPolyomaviridae dsDNA circular 5 Icosahedral NoPolyomaviridae dsDNA, circular 5 Icosahedral NoPapillomaviridae dsDNA, circular 7 to 8 Icosahedral No

ssDNAAnellovirus ssDNA circular 3 to 4 Isometric NoParvoviradae ssDNA, linear, (- or +/-) 5 Icosahedral No

RetroHepadnaviridae dsDNA (partial), circular 3 to 4 Icosahedral YesRetroviridae ssRNA (+), diploid 7 to 13 Spherical, rod or cone shaped Yes

dsRNAReoviridae dsRNA, segmented 19 to 32 Icosahedral No

ssRNA (-)Rhabdoviridae ssRNA (-) 11 to 15 Helical YesRhabdoviridae ssRNA (-) 11 to 15 Helical YesFiloviridae ssRNA (-) 19 Helical YesParamyxoviridae ssRNA (-) 10 to 15 Helical YesOrthomyxoviridae ssRNA (-), segmented 10 to 13.6 Helical YesBunyaviridae ssRNA (-, ambi), segmented 11 to 19 Helical YesArenaviridae ssRNA (-, ambi), segmented 11 Circular, nucleosomal YesDeltavirus ssRNA (-) circular 2 Spherical Yes

ssRNA (+)Picornaviridae ssRNA (+) 7 to 9 Icosahedral NoCalciviridae ssRNA (+) 7 to 8 Icosahedral NoHepevirus ssRNA (+) 7 Icosahedral NoAstroviridae ssRNA (+) 6 to 7 Isometric NoAstroviridae ssRNA (+) 6 to 7 Isometric NoCoronaviridae ssRNA (+) 28 to 31 Helical YesFlaviviridae ssRNA (+) 10 to 12 Spherical YesTogaviridae ssRNA (+) 11 to 12 Icosahedral Yes

ICOSAHEDRAL VIRUSES•1956 Watson and Crick only cubic symmetry•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 subunitssubunits

•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

b isubunits

Parvovirus Structure

Picornavirus Structure

QUASI-EQUIVALENCE1962 Caspar and Klug found a principal of building1962, 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 deformationp y g•Shell can contain 60n subunits

A Fuller geodesic domeThat inspired Caspar

d Kland Klug

Triangulation number (T) Enumerated by Caspar g ( ) y pand Klug

• T=f2 x P where f=# of subdivisions on each side of a triangular• T=f 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

O l T’ th t b d i d f th b ti• Only T’s that may be derived from the above equation are possible.

• 60 = minimal number of irregular subunits required• 60 = minimal number of irregular subunits required

CLASSES OF ICOSAHEDRAL DELTAHEDRA

Tabulation of the Triangulation Number TTabulation of the Triangulation Number T

Class

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

P = 3 3 12 27 . . . .

Skew Classes 7 13 19 21 . . . .

T Pf2 h P h 2 hk k 2 h d k i f i i hT = Pf2, where P = h 2 + hk + k 2, 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

CLASSES OF ICOSAHEDRAL DELTAHEDRA

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

Different Arrangements of Icosahedral Symmetry

Zlotnick A. PNAS 2004;101:15549-15550

©2004 by National Academy of Sciences

Jellyroll: Many but not all ViralJellyroll: Many, but not all Viral proteins

Capsid proteins

β-barrel.• Rhombohedral wedges:

– Fit into icosahedron.• Jellyroll topology

C d i ll• Conserved in many small viruses– T = 1 3T 1, 3, …

• 60, 180, 240 proteins…– RNA or DNA viruses.

E ti ll• Essentially no sequence homology.

Picornaviridae, a prototype T=3 virus• Quasi-equivalence with pentamer at each vertex andQuasi equivalence with pentamer at each vertex and

hexamers in other regions; • Triangulation # = 3.

N t th t VP 4 i t th f f th t t b t• Note that VP-4 is not on the surface of the structure but lies under the face.

Picornaviridae, a prototype T=3 virusThe protein subunits that form each protomer all assume a similar• 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”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.

TBSV icosahedron is 35.4 nm in diameterTomato Bushy Stunt Virus

• Each particle contains only a single molecule f RNA (4800 t) dof RNA (4800 nt) and

180 copies of the coat protein subunit (387 aa; 41 kd)

Vi i il t TBSVT= 3 Lattice

C• Viruses similar to TBSV will self-assemble in vitro from protein

C

psubunits + nucleic acid in an energy-independent fashion Protein Subunits C

N

independent fashion Protein Subunits Capsomeres

Assembly of Turnip Crinkle Virus (TBSV relative)HIV-1 Capsid

Scaffold-guided Assembly of Bacteriophage HK97

T=7420 subunits

Questions:

snekhai@howard.edu

Lecture Slides:

i kl ll h d d / h htwww.sicklecell.howard.edu/research.htm