RHINOVIRUS.ASCII

jean-yves sgro sgro at rhino.bocklabs.wisc.edu
Mon Feb 14 20:32:37 EST 1994


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                    A S C I I    R H I N O V I R U S 
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		      © 1994 Jean-Yves Sgro. 
    Institute for Molecular Virology- Madison Wi 53706 -USA-
                  sgro at rhino.bocklabs.wisc.edu





Published on the Internet. Reproduction granted for educational purposes. 
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(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)
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  (Note: Each section starts with @- for easy search on the computer)
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@- CLASSIFICATION:
-----------------

Rhinovirus is part of the Picornaviridae family. That is the *pico*rna*.
'PICO' means small, 'RNA' signifies that these are single standed positive 
sense RNA viruses. Thus these represent small RNA viruses.

The picornavirus family is divided in 5 Genera:

--------------------------------------------------------------------------
Genera		Serotypes	Members
--------------------------------------------------------------------------
-Enterovirus	    3		Human polioviruses 1-3
		    1		Human hepatitis virus A
		    1		Theiler's murine
					encephalomyelitis
		.........

-Rhinovirus       100		Human rhinoviruses 1-100
		    2		Bovine rhinoviruses 1-2

-Aphtovirus	    7		Foot-and-mouth disease virus 1-7

-Cardiovirus	    1		Encephalomyocarditis (EMC), Mengo

-Unassigned	   >3		Equine rhinoviruses 1-2, Cricket paralysis...
--------------------------------------------------------------------------


@- POLYPROTEINS:
---------------
The single RNA is translated into a polyprotein that is subsequently cleaved. 
The way the proteins cleave themselves out is slightly different amongst the
different genera. 
For our structural purposes suffice it to say that the coat proteins are
products of the cleavage of the P1 precursor protein. 

       <--------  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.


@- COAT PROTEINS:
----------------
In shematics and color graphics and movie representations I use the convention:

VP1 = blue
VP2 = green
VP3 = red
VP4 = yellow


Coat Protein Mass:
_______________________________________
| VP1	| VP2	| VP3	| VP4	| P1

32,300	28,500	26,200	7,200	94,200
_______________________________________

VP1-3 have 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 insersions 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 < depending on the position in 
the drawing. The Beta strands are labeled BIDG and CHEF. In the first plant 
virus solved an A Beta strand is found at the N terminus but is not preserved 
in other viruses.The loop names are also marked on the edges (BC,HI,DE,FG,CD,EF,
GH,EF).
The *** represent the 2 Alpha- helices. The helix between loop  EF and strand F
is in the back.

                        -
                       (   `-
                      ***    \    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. Itresults from the cleavage of VP0 into VP2 and VP4 which
occurs after assembly. In poliovirus the N-terminus is myristalated.



@- ICOSAHEDRAL CAPSID:
---------------------

60 of each of the coat proteins assemble into an icosymmetric structure 
with icosahedral symmetry. Rhinovirus has triangulation number of T=1 
but 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 whiles 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



@- CANYON and DRUG BINDING IN VP1 HYDROPHOBIC POCKET:
----------------------------------------------------

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 boud 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 Angstroems 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.


WIN 52035:
---------
  ___       ____          
 |   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:
------------------

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 resitant to a panel of murine monoclonal antibodies. "Mutations
were clustered in the serotype-variable regions of the amino acid sequences
fo the virus capsid proteins". These antigenic areas are found to protrude
out of the surface of the virus. Refer also to the above diagra, 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.




@- AVAILABLE 3D COORDINATES:
---------------------------

Rhinovirus has been crystalized as a native virus but also with
various drugs. The group from Michael Rossmann's at Purdue Univeristy
has released those structures to the protein databank (PDB) at brookhaven
which are available on anonymous ftp (pdb.pdb.bnl.gov [130.199.144.1]):

-------------------------------------------------------------------------------
PDB
ENTRY#	VIRUS					AUTHORS			  DATE
-------------------------------------------------------------------------------
1R1A  RHINOVIRUS 1A                             M.ROSSMANN ET AL.         12/88     
4RHV  RHINOVIRUS 14(HUMAN)                      E.ARNOLD,M.ROSSMANN        1/88     
2RS1  RHINOVIRUS/ANTIVIRAL AGENT 1S COMPLEX     M.ROSSMANN ET AL.         10/88     
2RR1  RHINOVIRUS/ANTIVIRAL AGENT 1R COMPLEX     M.ROSSMANN ET AL.         10/88     
2RM2  RHINOVIRUS/ANTIVIRAL AGENT 2 COMPLEX      M.ROSSMANN ET AL.         10/88     
2RS3  RHINOVIRUS/ANTIVIRAL AGENT 3S COMPLEX     M.ROSSMANN ET AL.         10/88     
2R04  RHINOVIRUS/ANTIVIRAL AGENT 4 COMPLEX      M.ROSSMANN ET AL.         10/88     
2RS5  RHINOVIRUS/ANTIVIRAL AGENT 5S COMPLEX     M.ROSSMANN ET AL.         10/88     
2R06  RHINOVIRUS/ANTIVIRAL AGENT 6 COMPLEX      M.ROSSMANN ET AL.         10/88     
2R07  RHINOVIRUS/ANTIVIRAL AGENT 7 COMPLEX      M.ROSSMANN ET AL.         10/88     
1R08  RHINOVIRUS/ANTIVIRAL AGENT 8 COMPLEX      M.ROSSMANN ET AL.         10/88     
1R09  RHINOVIRUS 14/R61837                      M.ROSSMANN ET AL.          5/90     
1RMU  RHINOVIRUS MUTANT((1)C199Y)               M.ROSSMANN ET AL.         10/88     
2RMU  RHINOVIRUS MUTANT((1)V188L)               M.ROSSMANN ET AL.         10/88   
1HRI  RHINOVIRUS 14(HUMAN)/ AGENT SCH 38057 	A.ZHANG,R.G.NANNI,E.ARNOLD10/92
-------------------------------------------------------------------------------
2PLV  POLIO VIRUS                               D.FILMAN,J.HOGLE          10/89     
2MEV  MENGO VIRUS                               M.ROSSMANN                 4/89   
-------------------------------------------------------------------------------



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@-



 		       		




 	       _________________________________________

	RHINOVIRUS QUICK-TIME 	M O V I E S  AVAILABLE VIA ANONYMOUS FTP on
 	       _________________________________________










			rhino.bocklabs.wisc.edu
			   [144.92.19.130]

___________________________________________________________________________

/pub/QuickTime-QuickPICS/Rhinovirus

 394752 Apr  8  1993 r14_em_QT.sea.MacBin
1041408 Apr  8  1993 r14_ico.sea.MacBin
1034368 May 13  1993 r14_nims.sea.MacBin
2634496 Apr  2  1993 r14_nims_autoplay_QuickPICS.MacBin
 682496 Jul  7  1993 r16_ico-2-Movie
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@------------------------------------------
 394752 Apr  8  1993 r14_em_QT.sea.MacBin
___________________________________________

Rhinovirus reconstructed as a 3D image from electron
microscopy microgrpahs of frozen-hydrated samples.
The microgrpahs were computerised and a 3D image
deduded from the various projections offered by the
set of 2D images.

While the virus is rotating it is possible to see the
5-fold and 3-fold axes quite clearly.

The resolution of the surface is at about 25 Angstrom
resolution.

@------------------------------------------
1041408 Apr  8  1993 r14_ico.sea.MacBin
___________________________________________

This series of images were actually my very first
quick time movie, made for an exhibit at the 
Science Museum and Industry in Chicago.

It represents rhinovirus as colored by its proteins.
VP1 is blue and sits around the icosahedral 5-fold axis
of symmetry, VP2 is green and VP3 is red.

@------------------------------------------
1034368 May 13  1993 r14_nims.sea.MacBin
2634496 Apr  2  1993 r14_nims_autoplay_QuickPICS.MacBin
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2 versions of the same movie. The 'autoplay' does not
require QuickTime but is bigger and less flexible to use.

Rhinovirus surface radially depth cued. The closer to the
surface the whiter the color, the lower the darker blue.

Antigenic sites have been colored in magenta. Antigenic sites
were determined by escape mutation of virus plated with various
antibodies.

@-------------------------------------------
 682496 Jul  7  1993 r16_ico-2-Movie
___________________________________________

Rhinovirus 16 instead of rhinovirus 14 but at this
resolution you can't tell the difference!

The structure of rhinovirus is shown with 2 pentamers
missing so as to see the VP4 protein on the interior.

------------------------------------------



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