__________________________________________________________________________
(For a more General Reference: Picornaviridae and Their Replication, CHAPTER 20. Roland R. Rueckert. Virology. Second Edition, edited by B.N. Fields, D.M. Knipe et al. Raven Press, Ltd., New-York - 1990)
This has been modified from the version found on the Wisconsin Virology WWW server (without permission...!) ___________________________________________________________________________
The Picornaviridae family: *pico*rna*. 'PICO' means small, 'RNA' signifies that these are single standed positive sense RNA viruses..
The picornavirus family is divided in 5 Genera and further subdivided into members.
This is an image reconstruction from X-ray crystallographic data of the r16 common cold virus: this is a member of the Rhinovirus genus of the Picornaviridae. Click on picture, then on the ">>" of the AAPLAY control line to animate: this will display the inner and outer capsid structures. VP1 is blue and clustered around the twelve 5-fold rotational axes; VP2 and 3 are green and red respectively, and cluster around the twenty local 6-fold rotational axes. VP4 can only be seen internally, where it clusters around 5-fold axes. Considerably more detail on structure is available below.
Picornaviruses also have a very similar genomic organisation to comoviruses - despite the latter having two genomic components instead of one - and can feasibly proposed to be evolutionarily related to comoviruses, albeit distantly. Still more distant is a relationship with potyviruses: these share only a "core" replicase-related sequence, and have filamentous particles rather than spherical.
<-------- P1>
---------------------------------------------------------------
Vpg---| VP0 | VP3 | VP1 |2A| 2B | 2C |3A|3B| 3C | 3D |
---------------------------------------------------------------
P1 - > VP0 + VP3 + VP1 and then VP0 -> VP2 + VP4 once the virus has assembled.
Vpg (virion protein genome) is attached to the 5'end of the RNA.
In schematics and color graphics and movie representations the convention:
is used.
_______________________________________ | VP1 | VP2 | VP3 | VP4 | P1 32,300 28,500 26,200 7,200 94,200 _______________________________________
VP1-3 has the now famous wedge-like protein fold motif shared by most virus structures solved by X-ray crystallography. It is created by a common CORE made up of 8 antiparallel Beta-strands forming a Beta-barrel structure. Proteins amongst viruses mainly differ in the loop insertions between the strands. These insertions accounts for a lot of the virus external appearance and also are the target structures for antibodies in the case of an animal virus. (see below). Interestingly there is no sequence conservation in the Beta strands structures of viruses although the structure is very well conserved.
Here is a representation of the 8-stranded Beta barrel. The beta strands orientation is
marked with the sybols: V,^,> and
-
( `-
*** \ EF
___>____C__ *** \
BC ( \ *** \
\ |_______ |
_\<_____h _________|_____________ HI ( \ *** | ' \ _\__\____E________>_|_/ \_
\ \ \ *** | COOH--\ \ GH
DE \ \ \ | / \ \
,_\_\<_\__f___/ / \ | FG \ \ \ \ |_************* \ | V ^ V ^ *************) | | G D I B | | | \ \ \ \ / | | \_\__\__\_______________>---/--/---'
\ \ \ / /
CD \__\__\_____________<_/ / \ \ / \__\____________>_/
\
|
|
NH2
The VP4 protein is much shorter and is found only on the interior surface of the virus. It results from the cleavage of VP0 into VP2 and VP4 which occurs after assembly. In poliovirus the N-terminus is myristylated.
60 of each of the coat proteins assemble into an icosymmetric structure with icosahedral symmetry. Picornaviruses have a triangulation number of T=1 but this is also refered to as a P=3 for Pseudo-T=3 structure. A T=3 structure occurs when all 180 (3 protein for each of the 60 facets) are chemically identical but have to assume slightly different neighbouring environment: The proteins which are around the 5-fold axis have 4 neighbours while the ones at the 3-fold axis have 5 neighbours.
___________________
/\ /\
/ \ / \
/ \ / \
/ \ / \
/ \ / \ T=1:
/ - V - \ A 20 facets
/ _ / /|\ \ _ \ Icosahedron.
/ _ / / | \ \ _ \ Contains
/ _ / / 60 \ \ _ \ 5-fold vertices.
/_ - /___|___\ - _\ T=3:
\ - _ | 20 _ - / Each of the 20
\ \ _ | - / / facets can be
\ \ _ | - / / subdivided into
\ \ _ ^ _ / / 60 total triangles.
\ | / Each of these 60
\ / \ / triangles contains
\ / \ / approximately one
\ / \ / each of the VP coat proteins.
\ / \ /
\/_________________\/
VP1 | VP1
5 Within each of the 60
. '/ \` . triangles fits about 1 each of
, / \ ` . VP1-3. The symmetry axes are
, VP1 / \ VP1 ` . \ labeled 5,2,3. p3 is a pseudo
, / VP1 \ ` ` . \ 3-fold axis within the triangle.
, / \ ` ` .\ Around the icosahedral
/ / \ ` VP3 ' 5-fold are five VP1s.
/ / \ `. . ./ But around the 3-fold
/ / \ / are 3 VP2s and 3 VP3s
VP3 /_______p3/ \ / / alternating.
/ \ \ / In Plant viruses with T=3
\ / \ VP3 \ VP2 / symmetry the similarity is
/ VP2 \ \ with VP1=A, VP2=C, VP3=B.
\ / \ \ / VP3
VP2__3/___________2_____\_________\3 __
\ |
\ \ / \ VP2
VP2 \ VP3 \ VP2 / \
\ \ / VP3 \
\ \_________/
\ / /
/
In T=3 plant viruses each protein is synthezised independently while in the polioviruses they arise from the cleavage of P1 as illustrated above. It is worh noting that the VP proteins issued from a single P1 precursor do not form a triangular face as the previous drawing might suggest. Instead they form what is usually refered to as the
*Biological* asymmetric
5 unit as opposed to the *crystallographic*
/ \ asymmetric unit. An asymmetric unit is
/ \ a set of minimum non-redundant information
/ \ necessary to reconstruct an icosaheron from
/ \ it using only the icosahedral symmetry.
/ \ The complete icosahedron is indeed
_______/ VP1 / mathematically obtained wether one uses
/ / / a triangular asymmetric unit or this
/ / / biological unit in which the VP3 area has only
/ /_______ / been switched to the right!
/ VP3 / \
/ / \
\ / VP2 \
\ / \
\3/_________________\
2
The VP1 proteins form a small cylindrical protrusion at the 5-fold axis. Around that protrusion is a depression sometimes refered to as the 'canyon'. This canyon is the receiving site for the cellular receptor which has been co-crystallized in this position by the crystallographers at Purdue University, and also observed there by cryo-EM reconstruction.
Here is a projection of the canyon onto one of the 60 triangular facets. The scales to the left shows the position in Angstroms along the X axis and range from 4 to 42. The bottom scale shows the size in Angstroms for the Y axis ranging from 01 at the right to 61 at the left; (the axis choice was slected by the crystallographers). The canyon appears as numbers within a background of + symbols. Numbers range from 1 to 4, 4 showing the deepest part of the canyon. The rim of the canyon is arbitrarily set at 137 Angstroems from the center of the particle. The output was prepared by the program V-suf (Rossmann and Palmenberg (1988), Virology 164,373-382)
X 5-fold
*
42 1.1
40 +++++++
38 1++++++++
36 +1+++++++1+
34 ++++++1++++++++
32 +1++++++++++++++
30 +11++1+++++++++++
28 12253++11112++++++++.
26 ++++++3433311+++++++1++
24 +++++++++++311+++++++3+++
22 ++++++++++++++3233222232111+++
20 +++++++++++++++22233443333331++++
18 +++++++++++++++++++33222334++++++++
16 ++++++++++++++++()++12111122++++++++++
14 ++++++++++++++++++++++++1++++++++++++++
12 .+1++++++++++++++++++++21+++++++++++++++.
10 ++++++++++++++++++++++++1++++++++++++++++++++
8 +++++++++++++++++++++++++++++++++++++++++++++++
6 +++++++++++++++++++++++++++++++++++++++++++++++++
4 +++++++++++++++++++++++++++++++++++++++++++++++++++ 3-fold
__________________________________________________________________
6655555555554444444444333333333322222222221111111111000000000 - Y values
1098765432109876543210987654321098765432109876543210987654321
The VP1 protein has also the peculiarity of having a hydrophobic pocket accessible from the surface via a small 'pore' entrance. It is indicated by the symbol () in the above drawing (approximate position). In some of the virus structures resolved by X-ray crystallography a substance can be found in this hydrophobic cavity. The nature of the compound is not known. It is refered to as a Sphingolipid in the case of poliovirus and as a sugar in the case of rhinovirus 1a, but crystallographers only see some 'extra' electron density. The pharmaceutical company Sterling Winthrop has synthezised compounds, often refered to as WIN-drugs, that diffuse readily in the VP1 pocket. (see e.g. Heinz et al. J. Virol. (1989) vol.63, pp 2476-2485, Genetic and Molecular Analyses of Spontaneous Mutants of Human Rhinovirus14 That Are Resistant to an Antiviral Compund). Other companies also study similar compounds (Jansen, Chalone, Sandoz) but none of them are ready for commercial use.
___ ____ | N / \ O____N | \\__<()>-O-- /\ __/ || |___O/ \____/ \/ \/ \\___||__CH3 Oxazoline Phenoxy Aliphatic Isoxazole Chain
The effect of the compound is to raise the canyon floor and prevent attachement to the cellular receptor that now cannot fit properly in the canyon and / or impair the uncoating of the capsid upon cell entry. The presence of such a compound in the structure confers thermal stability to the virion.
It is suggested that this is a trigger mechanism that allows the virus to survive in vivo. For instance poliovirus is stable in acid conditions and survives inside the stomach while rhinovirus does not.
Antigenic sites have been determined by escape mutations
Rueckert et al. (1986), Virus Attachement and Entry Into Cells, American Society for Microbiology: Location of four neutralization antigens on the three- dimensioan; surface of a common-cold picornavirus, human rhinovirus14;
Rossmann et al. (1985), Nature, 317,145-153. Structure of the human common cold virus and functional relationship to other picornaviruses)
Viruses were resistant to a panel of murine monoclonal antibodies. "Mutations were clustered in the serotype-variable regions of the amino acid sequences for the virus capsid proteins". These antigenic areas are found to protrude out of the surface of the virus. Refer also to the above diagram, showing the depth of the canyon...
Four neutralization Immunogens (NIm) sites have been resolved by cross-neutralization tests. Sites are labeled NIm IA, IB, II, and III. The ** symbols approximate the size of the area. IB is a smaller area than IA.
5
/ \ IA::(VP1): Q83,K85,D138,S139
/ IA\ IB::(VP1):D91,E95
/ ** \
/ \ II::(VP2):E136,S158,A159,E161,V162
/ IB \ (VP1):E210
/ / \
/ / \ III::(VP3):N72,R75,E78,G203
/ / \ (VP1):K287
/____*__ / \
/ ** * \ \
/ II ** \ * \
/ \ III* \ The major antigenic site is on the BC
3/________________\_________\3 loop in VP1 and VP3 but in the EF loop
2 in VP2.