Viral Replication for Beginners

[mixin]

This is something I've compiled from a number of sources. I pulled some of it from Vincent Racaniello's blog so I need to give credit. Some of it is just my understanding so if anyone sees something that is incorrect, please let me know.
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Flu Basics
Influenza generally prefers the upper respiratory tract, which consists of the nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi. The pandemic H1N1 is occassionally infecting the lower tract also.

Within nasal secretions, millions of virus particles per ml are shed, so that a 0.1 µl aerosol particle contains more than 100 virus particles. A single HID (human infectious dose) of influenza virus might be between 100 and 1,000 particles.

Particles are usually grouped by sizes, which help dictate how far down the respiratory tract they can go.
Particles in general (subbing um for micrometer):
Range in size from 0.1 um to over 1000 um
A single particle of 100 um will contain 1000 times more virus than a single 10 um particle
2 to 24 um particles accounted for 90% of the number of particles emitted but a small fraction of the total volume
Viral concentration will vary among persons and the course of their infection

Balistic Particles:
Greater in size than 100 um fall out of the air in seconds and have a range of less than a metre
Can be deposited directly on mucus membranes
Have low probability of being inhaled

Inhalable Particles:
Range in size from 0.1 to 100 um
Particles near 100 um deposit exclusively in the nasopharynx
Nasopharyngeal-sized particles, from 20 to 100 um, tend to travel no further than upper respiratory tract
Tracheobronchial-sized, from 10 to 20 um, may deposit as far down as the tracheobronchial region
Alveolar-sized, less than 10 um, may deposit anywhere, but are the only size capable of reaching the alveolar region
Depending on size, can stay in the air from seconds to days
Air currents may carry them considerable distances
Smaller particles evaporate faster

 

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Re: Viral Replication for Beginners

Structure of influenza virus
The influenza virion (as the infectious particle is called) is roughly spherical. It is an enveloped virus – which means the outer layer is a lipid membrane which is taken from the host cell in which the virus multiplies. Protein ’spikes’, known as HA (hemagglutinin) and NA (neuraminidase) are inserted into the lipid membrane. The HA and NA are important in the immune response against the virus; antibodies (proteins made by us to combat infection) against these spikes may protect against infection.. Also embedded in the lipid membrane is the M2 protein, which is the target of the antiviral adamantanes – amantadine and rimantadine. The NA protein is the target of the antiviral drugs Relenza and Tamiflu

Beneath the lipid membrane is a viral protein called M1, or matrix protein. This protein, which forms a shell, gives strength and rigidity to the lipid envelope. Within the interior of the virion are the viral RNAs – 8 of them for influenza A viruses. These are the genetic material of the virus; they code for one or two proteins. Each RNA segment, as they are called, consists of RNA joined with several proteins shown in the diagram: PB1, PB2, PA, NP. These RNA segments are the genes of influenza virus. The interior of the virion also contains another protein called NEP.

 

[mixin]

Re: Viral Replication for Beginners

Protein Functions
Each of the proteins has a primary function:
M1 forms the shell (matrix) of the virus particle
HA attaches the virion to the sialic acid on the host cell (produces the protein that recognizes the host cell receptor)
M2 acts as an ion channel pump (in the lipid layer) to lower or maintain the pH of the endosome
NS1, NS2 act as toxins to promote the production of the virus in the infected cell
PB1, PB2, PA form the active RNA-RNA polymerase
RNA-RNA polymerase, which is responsible for replication and transcription
NA, an enzyme, cuts (cleaves) the binding sites on the host cell, releasing the new viruses. It also cleaves sialic acid residues from viral proteins, preventing aggregation of the new viruses.


Influenza virus attachment to cells
Viruses cannot reproduce outside of a cell. The production of new infectious particles must take place within a cell. Upon entering cells, viruses parasitize the host machinery to produce new viral progeny. The sum total of all the events that take place in a virus-infected cell is called the infectious cycle, or viral replication.
Here is a typical cell.



In order to enter a host cell, the virions must attach to a receptor on the plasma membrane. Every virus has a specific receptor that it attaches to (see the coated pit in the above picture), and in turn there is a particular viral protein that binds this cell receptor. Here is an illustration of an influenza virion binding to its cell receptor



You can see the individual HA ’spikes’ on the virion binding to a structure on the cell. The cell receptor is sialic acid – a small sugar that is attached to many different proteins on the cell surface. Here’s what sialic acid looks like.



On the left is a drawing of a cell protein embedded in the plasma membrane. The interior of the cell – cytoplasm – is at the bottom. Part of the protein (the green part) crosses the membrane, and there are also parts on the cytoplasmic and extracellular sides. The spheres are sugars that are attached to many proteins (protein + sugar = glycoprotein). Sialic acid is always the last sugar in a chain (see the red arrows) that is attached to a protein. . Influenza virions attach to cells when the HA grabs onto the very small sialic acid On the right is the chemical structure of sialic acid; the next sugar, to the right, is galactose.

The sugar is actually quite tiny compared to the HA – it fits into a small pocket on the top of the spike. Here is a molecular model showing the HA bound to an analog of sialic acid. The globular top of the HA is at the top of the image. The tiny red and white spheres show where sialic acid would be bound, in a pocket at the top of the HA.

 

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Re: Viral Replication for Beginners

Release of influenza viral RNAs into cells



After the virion attaches to the receptors at the cell surface, the virus-receptor complex is taken into cells by endocytosis, a process by which cells normally take up molecules from the extracellular fluid (it pulls the attached particle inward and then surrounds it).

As the endosomal vesicles that contain the virus particles move towards the cell nucleus, their pH drops. This change in pH is accomplished by a protein in the virion membrane called M2. This viral protein forms a channel in the membrane that actively pumps protons from the endosome into the interior of the virion. These protons lower the pH in the interior of the virion, releasing the viral RNAs from the M1 and into the cytoplasm (the unzipping of the coat). They are then transported into the cell nucleus where viral RNA replication occurs. Replication is quick; after only 6 hours, the first viruses are shed from infected cells.

The M2 ion channel is the target of the antiviral adamantanes, depicted below. These compounds clog the channel and prevent it from pumping protons into the virion. In the presence of adamantanes, viral RNAs remain bound to M1 and cannot enter the nucleus. Therefore viral replication is inhibited. Resistance to adamantanes occurs by changes in amino acids that line the M2 channel. These changes prevent the drug from plugging the channel.

 

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Re: Viral Replication for Beginners

Neuraminidase Release of the New Virions
As we can see in this picture, the new virion is still bound to the infected host cell. It’s the job of NA to cleave the sialic acid residues from the cell surface, which releases the new virus to go on and find a new host. Removing the sugars from the newly budded virions also prevents them from attaching to each other.




Influenza HA cleavage is required for infectivity
Note: this cleavage is different from the NA cleavage of the new virions from the infected host cell.

The influenza virus hemagglutinin (HA) is the viral protein that attaches to cell receptors. The HA also plays an important role in the release of the viral RNA into the cell, by causing fusion of viral and cellular membranes. HA must be cleaved by cellular proteases to be active as a fusion protein.

The HA on the influenza virion is a trimer: it is made up of three copies of the HA polypeptide. The cleavage site for cell proteases on the HA protein is located near the viral membrane.



In the diagram, the globular head of the HA protein, which attaches to cell receptors, is at the top, and the viral membrane is at the bottom. For clarity, only one HA cleavage site is labeled. The uncleaved form of the protein is called HA0; after cleavage by a cellular enzyme, two proteins are produced, called HA1 (blue) and HA2 (red). The two subunits remain together at the surface of the virus particle. The new amino(N)-terminal end of HA2 that is produced by cleavage contains a sequence of hydrophobic amino acids called a fusion peptide. During entry of influenza virus into cells, the fusion peptide inserts into the endosomal membrane and causes fusion of the viral and cell membranes. Consequently, the influenza viral RNAs can enter the cytoplasm. The fusion process is described in a previous post.

If the HA protein is not cleaved to form HA1 and HA2, fusion of the new virion to the new host cell cannot occur. Therefore influenza viruses with uncleaved HA are not infectious. Cleavage of the viral HA occurs after newly synthesized virions are released from cells.

 

[mixin]

Re: Viral Replication for Beginners

If anyone has anything to add, I'll be glad to edit it in.

 

[gsgs]

Re: Viral Replication for Beginners

H5 and H7 occur in high-pathogenic and low-pathogenic forms.

High pathogenic can develope from low pathogenic by mutation
in poultry. It just contains additional amino-acids on
the HA-cleavage site which generally make it more
pathogenic. Presumably by allowing it to be cleaved in
more sorts of cells.
Apparantly this presents no additional evolutionary advantage
in wild birds, so presumably those cells are not useful
for virus transmission in wild birds.

 

[mixin]

Re: Viral Replication for Beginners

Thanks to Gs for the additional information.

This is from JJackson as he helped me further understand the role of NA cleavage: :tiphat:

It is confusing because sialic acid residues are very common. They are found on both the outside of the virus and on the outside of the host cell. The key points are NA is a specialised sialic acid cleaver. When the virus binds to a cell (step 1) it needs a host cell receptor to bind to - as its NAs are removing these it will make it harder for the cell to bind - but once binding occurs then the HA must be cleaved by a host cell protease (the virus does not bring along its own scissors for this job). Cleavage - of the HA - then starts the fusion of virus and host cell - along with the ion pump and other processes. Once in the cell new virions are assembled and are released by budding. Now the virus has a problem it is covered by HAs which - given half a chance - will bind to sialic acids on the surface of the cell it is trying to escape from. This is not what it wants it wants to get away from the cell that made it and find a new cell to infect so it needs its NAs to cut off all the sialic acid residues so it can drift off and find fresh cells to infect. It also wants its NAs to get rid of any sialic acid residues on itself or other virus particles will bind to it rather than to cells they can infect.

The confusion is that the binding site (on the HA) attaches to the receptor (a terminal sugar on a host glycoprotein) the NA cuts off these sugars which stops the virus from binding. This is actually a bad thing for the virus when it is trying to infect the cell but a very good thing for the virus after budding when it wants to go off and find new cells to infect. Being able to make a clean get away at budding outweighs the disadvantages of NA interfering with infection.

When we talk about NA cleavage NA is the thing doing the cleaving (it is pruning sugars of a glycoprotein) but when we talk about HA cleavage it is the HA that is being pruned (not doing the pruning), in this case by a protease (a protein that cuts other proteins) made by the infected organism.

 

[Marsey]

Re: Viral Replication for Beginners

Thank you so much! I did very well till I got to HA cleavage and then my brain started to hurt. You've done a great job and the pictures are very helpful. I will come back to it later and see if I can get the last part.

 

[sharon sanders]

Re: Viral Replication for Beginners

Thank you so much! I did very well till I got to HA cleavage and then my brain started to hurt. You've done a great job and the pictures are very helpful. I will come back to it later and see if I can get the last part.

Ditto!

 

[mixin]

Re: Viral Replication for Beginners

Adsorption and penetration
The virus adsorbs to receptors on the cell surface and is internalized by endocytosis. At acid pH of an endosome, HA undergoes a conformational change and fusion occurs. Nucleocapsids are released to cytoplasm.

Transcription, translation and replication
Nucleocapsids are transported into the nucleus. mRNA synthesis and replication of viral RNA occurs in the nucleus. This is very unusual for an RNA virus. Influenza virus has an unusual mechanism for acquiring a methylated, capped 5'end to its mRNAs.

A viral endonuclease (which is packaged in the influenza virus) snips off the 5'end of a host capped, methylated mRNA about 13-15 bases from the 5' end and uses this as a primer for viral mRNA synthesis (figure 20) - hence all flu mRNAs have a short stretch at the 5' end which is derived from host mRNA.

The viral RNA polymerase (transcriptase) extends the primer and copies the template into complementary plus sense mRNA and adds a poly(A) tail. Transcription results in 8 primary transcripts, one transcript per segment. Some segments give rise to primary transcripts which can be alternatively spliced (since influenza virus RNA synthesis occurs in the nucleus, it has access to splicing machinery), each giving rise to two alternative transcripts. For example, the M segment gives rise to two alternative mRNAs. These code for the M1 protein and the M2 protein. Thus a single segment can code for more than one protein since the virus has access to splicing machinery. The mRNAs are translated in the cytoplasm. Transmembrane proteins are moved to the plasma membrane while proteins needed for RNA replication are transported to the nucleus.

Replication of RNA
RNA replication occurs in the nucleus using a virus-coded enzyme (this may be same as the RNA polymerase involved in transcription of mRNAs, or a modified version). A full length, exact complementary copy of virion RNA is made - this plus sense RNA is probably coated with nucleocapsid protein as it is made. Full length plus strand RNA is then used as a template for full-length minus strand synthesis; again the new minus strand is probably coated with nucleocapsid protein as it is made. New minus strands can be used as templates for replication, mRNA synthesis, or packaged.

Assembly
This occurs at the plasma membrane. Nucleocapsids are transported out of the nucleus while envelope proteins are transported via the Golgi body to the plasma membrane. The M1 protein interacts with both nucleocapsid and a modified region of the plasma membrane which contains the glycoproteins HA and NA. Virus then buds out through the host cell membrane.

NOTE:
HA needs to be cleaved before it can promote fusion. Cleavage occurs as the virus leaves the cell or in the extracellular fluid. The requirement for cleavage affects which tissues can produce infectious virus. The cleaved protein needs to then undergo a conformational change, usually caused by exposure to a acidic endosome environment when it infects the next cell, before it can cause fusion.

NA probably helps the virus leave the cell by removing sialic acid from receptors. NA may also help the virus penetrate mucus to reach epithelial cells of the respiratory tract by enabling it to dissociate from sialic acid-containing receptors in the mucus by destroying them. The neuraminidase does not prevent the virus infecting new cells because endocytosis is presumably faster than receptor removal.

http://pathmicro.med.sc.edu/mhunt/RNA-HO.htm