VIRUS STRUCTURE



OVERVIEW

Why do we care about the structure of a virus? Knowing the structure of a virus gives hints about how the virus interacts with the cell to initiate the infectious process. The structure also gives hints about epitopes that interact with the immune system. From this information, strategies to abort virus infection might be devised.


During one of these class meetings, we will see a videotape about the structure of poliovirus. Unfortunately, there is a lot of information packed into this tape and it goes by pretty quickly. Things to try to "grab" are the axes of symmetry, the arrangement of the protomers, the relationship of icosahedral viruses that infect different hosts, and the function of the loops.



HOW VIRUS STRUCTURE IS STUDIED

Virus structure has been deciphered in two ways: electron microscopy and X-ray diffraction. Electron microscopy at low magnification can show how the virus interacts with the cell. This is a micrograph of bacteriophage T4 at the surface of a cell (Fig.7/8.1).
At high magnification, electron microscopy gives a picture of the surface of the virus. The micrograph below is of adenovirus (Fig.7/8.2). I hope you can see the regularity on the surface and the little rods and balls protruding from the surface.
We can also get good pictures of enveloped viruses. Below are microgrphs of influenza virus (Fig. 7/8.3a.) and of a coronavirus (Fig.7/8.3b). The peplomers in the envelopes of both these viruses are visible.

X-ray diffraction requires that the virus be crystallized before subjected to the x-ray beam, often a difficult task. The crystalline virus diffracts or bends the x-rays; the position of the diffracted rays is captured on film and sophisticated analysis, that used to be vary laborious but is now done easily on computers, gives information about the structure of the virus. The image below is a computer reconstruction of the surface of adenovirus (Fig. 7/8.4).
At higher resolution, x-ray crystallography can "place" every atom within a viral protein (Fig. 7/8.5).


In the past few years, the technique of x-ray crystallography has been combined with molecular biological techniques for the expression of large amounts of single proteins or portion thereof. Having large abouts of these peptides means that crystals can be grown easily and quickly. Sometimes portions of two proteins are co-crystallized to study how they interact.

TWO TYPES OF VIRUS STRUCTURE: HELIX AND ICOSAHEDRAL

How do you think the structure of a virus is organized? What components are on the outside and which ones are on the inside? What is the advantage to the virus of this organization?

What general principles explain the structure of viruses? First, viruses come in two basic shapes, helical and icosahedral. (For enveloped viruses, these shapes are those of the nucleoprotein core within the envelope.) TMV, our old friend, is the prototypical helical virus and poliovirus is an example of an icosahedral virus (Fig. 7/8.6).


Helix

Helical viruses are defined by their pitch, the distance from one point on the surface of the virus to the point above or below it at which the protein of the coat is in exactly the same orientation. TMV has a single protein in its coat. In the helical arrangement, you have to follow the proteins three turns around the outside of the helix to get to a point where one protein is exactly above another. As you go around the helix in this manner, you would count 49 protein molecules, 16.3/turn. Each protein molecule interacts with 3 nucleotides in the single RNA molecule of the viral genome. The RNA itself is also arranged as a helix within the core of the virion.

Icosahedron
The icosahedron is a geometrical solid, the most recognizable version of which is a soccer ball. It has 12 vertices (points), 20 faces (flat sides), and 30 edges. Each vertex, face or edge is an axis of symmetry. Looking carefully at an axis of symmetry reveals that it is a place on the surface of the solid about which rotation of the figure will result in its looking just the same as before you began rotation (Fig. 7/8.7).

The number associated with an axis of symmetry (the n-fold axis) is the number that is equal to the number of positions during a 360 degree rotation at which what you’re looking at looks the same as the original position. What is the number for the "fold" when looking at the vertex, at the face, at the edge?

We need to be able to identify the face, vertices and edges, because these are the names that virologists use to describe positions on the virion. They "shorthand" and say things like "The virus interacts with the cell at the 5-fold axis." Also, the number of protein units found on the edge is the basis for the classification of viruses by the triangulation number or T. This is another shorthand term that encompasses how the proteins are arranged on the surface of the virus and how many protein units are on the surface.

This diagram (Fig. 7/8.8) represents schematically a virus of the T=4 type. You are looking at a face. Along the edge, there is one capsomer, that divides the edge into two segments. The T number is the number of segments between vertices squared.

What is the T number for this virus shown here?

An alternative way to get the T number is to connect all the capsomers with lines and count the number of triangles on the face.

Can you find the hexamers on this face?

CAPSOMERS AND PROTOMERS

Definition of terms

Protein units on the surface of a virus are as capsomers and or protomers. The basic structural unit of a virus’ coat is called a capsomer. Capsomers can be made up several proteins, each of which is a protomer. For viruses with only one protein in their capsids, the protomer and the capsomer are equivalent.

Another layer of descriptor refers to how the capsomers are arranged. A capsomer at the vertex is called a pentamer because it is within a unit of five capsomers. Capsomers on the face of a virus, if the virus is large and complex enough, are called hexamers, since they are within a recognizable hexamer, or group of six. Look back at Fig. 7/8.6 for a good graphic illustration.

Here is Fig. 7/8.4. Can you find the hexamers on adenovirus?

The significance of capsomers
Capsomers are the basic building blocks of viral architecture. The important concepts about capsomers are :

The first of these concepts agrees nicely with what we know about the chemistry of proteins. The capsid is assembled by protein/protein interaction. There are two ways for this assembly to occur. One is spontaneous interaction between the capsomers, when this is thermodynamically favorable. The other is an ordered sequence of events, with or without the intervention of cell-coded proteins. Once a capsomer is assembled, the formation of the capsid should be pretty easy, since the surfaces or edges of all of them have the same chemical group. Self-assembly would then be like putting together a simple jig-saw puzzle whose pieces have only a few shapes, e.g., for all of them opposite sides have "outies" and the other pair of sides have "innies".

The second of concepts ties in nicely with what we know about viral genomes. Viral genomes are usually small(ish). If there were many different protomers comprising the capsid, much of the coding capacity of the virus would be used up to provide these proteins. Here’s an example: the capsid of bovine parvovirus is made of three proteins of ~ 60, 70 and 80 kDa. The genome is 5517 nt, as its genome is single-stranded DNA. The only way to code for these proteins within the constraints of the genome size is to use a portion of the genome repetitively for the capsid proteins. (Can you do the computation to prove to yourself that this is true?) The way this difficulty is overcome is for all three BPV capsid proteins to have the same amino acid sequence for most of polypeptide(s). The proteins are a nested set: all of the coding sequences of the 60 kDa protein is virtually the same as that of the 70 dDa protein and all of the coding sequences of the 70 kDa protein is virtually the same as that of the 80 dDa protein. The three proteins differ from each other only at the amino termini (Fig. 7/8.9).


The structure of protomers

For icosahedral viruses, the determination of the three-dimensional structure of the protomers contributes to our understanding of virus structure and function.

With respect to structure, there are two major components to the shape of the protomers; the b-barrels (regions of protein structure called a b-pleated sheet) and the loops. The illustration below shows these two elements in capsid proteins VP1, VP2 and VP3 of poliovirus (Fig. 7/8.10).

The b-barrels are the straight lines with the arrows at the ends (the direction is from N-terminus to C-terminus). The loops are the irregularly shaped lines that connect the b-barrels.

Before you read the next paragraphs, try to answer this question: "What do you think is the function of each of these domains of the protein(s)?" I give you the information that the b-barrels are internal to the structure and relatively rigid while the loops are on the surface and more "floppy".

Let’s talk about the b-barrels first. These are the portions of the protomers that define the shape of the icosahedron. There are only so many different ways that a protein(s) can be constructed so that they fit together to create a capsid with a defined structure. Based on this information, compare the structure of the protomers of an icosdahedral virus that infects plants to one that infects animals.

The basic icosahedral structure of viruses can also contribute to theire ability to infect cells. In order for infection to occur, there had to be an interaction between the cell-coded protein that serves as the receptor for the virus and some component of the virion. Shown below is the structure of the vertex (5-fold axis of symmetry) of a picornavirus, the family of viruses that include Rhinovirus, the cause of the common cold and poliovirus (Fig. 7/8.11).

The structural feature at the vertex that is of importance in virus-receptor interaction is the "canyon", a depression around the 5-fold axis. An antibody molecule is shown in the diagram; what do you infer about the function of anti-viral antibodies from this diagram? How might knowing that the cellular receptor interacts with the rhinoviral "canyon" help design an anti-viral drug?

The loops that link the b-barrels is where another facet of the biology of viruses occurs. Since these loops are exposed on the surface of the virus, they are what the infected organism "sees" and responds to. Before you read the next paragraph, try to answer this question: "What system within an organism responds to the loops?"

OK - I’ll give you the answer to keep the discussion going - it’s the immune system. Antibodies are produced in response to the shapes assumed by the amino acid sequences found on the loops. These sites on the loops are called epitopes. The antibodies bind to the epitopes and mask them, thus inhibiting the replication process. This process is called neutralization, and antibodies that do this are called neutralizing antibodies. The antibodies produced to some viruses, such as HIV, are not very efficient at neutralization, allowing the virus to gain footholds in the organism. The location of antibodies on the surface of a virus has been confirmed by electron microscopy. Fig. 7/8.12 shows in white the location of antibodies on the surface of rhinovirus.

A fabulous experiment was recently done that ties together virus interaction with the surface of the cell and the position of antibodies on the surface of a virus. The virus involved is HIV, an enveloped virus. HIV initiates infection of cells of the immune system by the binding of a peplomer called gp120 to a cell surface molecule called CD4. (gp120 stands for a glycoprotein, a protein whose molecular weight is 120,000 daltons with sugar molecules attached.) A second event is necessary for the virus to enter the cell, and that is binding of gp120 to a second cell surface protein, CCR-5, that normally functions as a chemokine receptor. By X-ray diffraction at a resulution of 2.5 A, scientists solved the crystal structure of a complex of important portions of gp120, CD4 and an antibody that prevents interaction of gp120 with CCR-5. Their results show that after binding of gp120 to CD4, gp120 undergoes a conformational change that is necessary for its binding to CCR-5. During this change, new epitopes are formed that make up a new site that is required for binding to CCR-5. This new spatial arrangement of gp120 is not seen by the immune system, so antibodies are not produced that can block this step of HIV entry.


SUMMARY
The structure of viruses has been deciphered by electron microscopy and X-ray diffraction. These techniques identify two shapes, helical and icosahedral. Helical viruses are defined by their pitch and icosahedral viruses by their axes of symmetry; the 2-fold axis at an edge, the 3-fold axis on the face, and the 5-fold axis at a vertex. The number of types of proteins that make up a capsid are limited. Each individual protein found in a capsid is called a protomer. Protomers can be grouped into units called capsomers; these are joined by protein/protein interaction to form the shell of the virus with the nucleic acid on the interior. The arrangement of these capsomers on the surface allows classification of icoasaheldral viruses by their T or triangulation number. Proteins on the viral surface self-assemble or come together in a sequential fashion.

X-ray diffraction studies have elucidated the structure of individual protomers. For icosahedral viruses, these proteins contain two domains, b-barrels and the loops that connect them. This arrangement is common in icoashadral viruses irrespective of the nature of the host. The b-barrels are responsible for the shape of the virus while the loops contain the biological determinants, the epitopes that interact with the immune system. For some viruses, the point on the virus that interacts with the cell has been identified: for picornaviruses, the receptor interacts at the vertex; for HIV, gp120 interacts first with the cell surface CD4 molecule, undergoes a change in conformation and then interacts with a second receptor, the cell surface CCR-5 chemokine receptor.

Knowing the structure of a virus gives hints about how the virus interacts with the cell to initiate the infectious process and how the structure of the viral proteins allows interaction with the immune system. From this information, strategies to abort virus infection might be devised.