Fun II: 11:00 - 12:00Scribe: Hillary Carney

Wednesday, November 4, 2009Proof: Jennifer Grimes

Dr. MooreIntroduction to VirologyPage1 of 8

Introduction [S1]:
We’re going to start today with a lecture on viruses. I’m going to give you some background on viruses. Tomorrow we will look at virus families, + and – RNA viruses, etc.
We’re going to talk about a couple of things today. Viral replication, which is how viruses exploit cells to make new viruses and viral pathogenesis—that is how viruses cause disease and how viruses escape and interact with the immune system.
You’re responsible for what he put on the slides. There’s no real reason to memorize everything on the slides. He will tell us what we don’t have to memorize.
The DNA/RNA Protein Pathway [S2]
In order to understand viral replication and viral pathogenesis we need to talk about the central dogma of molecular biology: DNA to RNA to protein.
Viruses can be either DNA or RNA, so if you recall your cellular DNA is transcribed mainly by an enzyme called RNA polymerase to make messenger RNA. Then the messenger RNA is then translated by cellular proteins and other RNAs to make cellular proteins, and this is how a cell lives and survives.
Viruses can either be DNA or RNA, but in order for a virus to work it has to utilize the host cell translational machinery. Viruses are too small to encode all of the genes for translation. So when they come into a cell they are going to usurp this translational machinery, many times they are going to usurp the DNA and RNA metabolism.

Characteristics of Viruses [S3]
So what are the characteristics of viruses?
They are filterable agents. Means that they pass through filters that can capture bacteria. The person who found thins was Pastuer. When he would filter his bacteria and he had a clear filtrate that if he put that in with bacteria it would kill that bacteria.
It’s important because it tells you that viruses are small.
Viruses are obligate intracellular parasites. They need cells to divide and replicate. They need cells to make new viruses. You can’t see free virus replicating in a tube. They can’t replicate unless they have cells.
Viruses have minimal genetic information. They are very efficient. From what we know about molecular biology, a lot of cell biology, a lot cell biology, was first understood with viruses because we could understand viruses. This is because they’re small enough to sequence and understand the importance of viral gene expression.
They rely on the host cell machinery to fulfill the replication cycle.
They are generally assembled from building blocks encoded by the virus. Don’t divide like cells.We’ll see that when we look at some of the growth curves for viruses.
Absolutely require host cells for replicate to produce proteins needed to synthesize new viral genomes, the building blocks of virus structure. So once again, viruses don not replicate outside of cells.

For a Virus to be successful [S4]
For a virus to be successful it has to be able to be transmitted through potentially harsh conditions. Important when you get into your practices, because that is where viruses like to get transmitted—through the mouth, through the eye—and for sexually transmitted viruses also.
Some of these viruses are stable in very harsh conditions.
Core viruses—which are stable in your gut—and they are excreted into the environment and then transmitted in a fecal oral fashion. Other viruses are stable in the air—like the flu virus. This is why you see people walking around with masks on all the time to keep from getting H1N1. This is because these viruses are stable in aerosolization.
Most viruses traverse the skin or other barriers of the host. Your most effective defense against a virus is your skin. It’s the largest organ and it is very effective at preventing viruses from entering the body.
Must adapt to the biochemical machinery of the host cell for replication.
What this means is that these virus will take over the host machinery for replication so they will make more viruses and they have devised numerous ways to do this.
They have also found ways to escape the host immune response. So whether or not you realize it you’re at constant war with the environment and with viruses. You immune system is monitoring itself, looking for viruses, and it’s going to response to viruses and those viruses are trying to avoid the immune response. If you win that battle you come to school tomorrow. If you lose that battle you’re in bed sick tomorrow.
Basic components of a virion [S5]
DNA or RNA
Structural proteins—which are capsid proteins. These surround the DNA and the RNA. This protects the virus from the environment.
Sometimes within these capsids you have enzymes and nucleic acid binding proteins to help the virus along. This can either form a nuclear capsid or a naked capsid virus.
The nuclear capsid virus most of the time is surrounded by a membrane, which embedded in this membrane are glycoproteins. This is called an enveloped virus. The best way to visualize these is that these membranes are like soap bubbles, with the nucleic acid inside. That’s how the virus has evolved and how it’s transmitted to host cells.
Virus classification [S6]
Various ways to classify viruses.
Size, morphology, genome type (DNA or RNA), and means of replication.
There’s DNA viruses, envelopedvirues- surrounded by membrane. There are pox virus (small pox virus), herpes virus, and hepadno virus, and these are all mobile.
There are naked capsid- palyomo viruses, adeno viruses, as well as papilloma viruses—those are DNA viruses—means that genetic information is encoded by DNA.
There are many more RNA viruses. These are viruses where the genetic information inside the environ is RNA.
We’ll talk more tomorrow about all of these. The noro virus is what you get on cruise ships.
Negative sense RNA viruses.
Remember that RNA is single stranded, so you can have two senses. These viruses include a lot that you hear about today.
Orthomyxo viruses—that’s flu virus.
Filo viruses—that’s ebola viruses as well as numerous other exotic viruses.
There are also double stranded viruses. Both + and – RNA. These are reo viruses and gastrointestinal viruses.
In the far corner of the chart is a virus that is actually a plus strand RNA virus. This is a retrovirus –starts out as RNA but then goes back to make DNA, integrates into your chromosome, to make new virus. This is HIV.

Relative sizes of viruses [S7]
This is E. coli
One of the largest is the Pox virus. If you have very good eyes and you’ve had a few drinks you can see these using a light microscope. He’s never seen them in a light microscope.
Highlights that all these other viruses, which run the gamut, and these are viruses that infect E. coli (called bacteriophages), but these are so small that you have to use an electron microscope to see these viruses. That’s an important point in terms of size. Viruses are not bigger than the cells they infect.

General structure of viruses [S8]
There are two general structures of the virus: Naked capsid and nucleic capside.
Naked capsid—is just a capsid surrounding a nucleic acid.
Nucleic capsid- this is the viral genome, this is an RNA genome, in which proteins are sticking to it.
The Difference is that that the nucleic protects the virus from the environment and the naked doesn’t. Both of these can be enveloped, meaning that can have a lipid bilayer --that soap bubble. In the middle of this “soap bubble” is this icosahedra virus. Embedded on the outer surface of this envelope are what we call glycoproteins and structural proteins sticking outside of the virus. This is what your immune system sees. Most of the nucleic capsid structures are in enveloped viruses that protects the virus from the environment. This is analogous to what a flu virus would look like.

Genome contents of viruses [S9]
The genome contents of viruses can either be RNA or DNA. The RNA viruses can be single, double, liner, or segmented. DNA can be double, linear, single, circular.
Don’t have to memorize this.
What he wants us to get out of this slide is that viruses can come in all shapes and sizes as well as having different genomes—DNA/ RNA, single stranded/double stranded.

Naked vs. Enveloped Viruses [S10]
This translates into your practice when you are trying to prevent these viruses from coming into your practice or trying to get rid of them by sterilizing.
Naked capsids—this is a proteinase structure that is very tight, analogous to a little b.b. surrounding a nucleic acid. They can withstand very harsh environmental conditions. They are resistant to drying, acids, and detergents. Many are transmitted by fecal-oral route. Lysol doesn’t kill these viruses. Naked are the type you’ve probably heard about— the Human Rhinovirus. This will sit on your counters and on your instruments. It’s a very stable virus.
In contrast, enveloped viruses—like the flu virus—have a lipid membrane surrounding it. So how do you get rid of soap bubbles? You pop them. Soap and detergent will pop a soap bubble. You can’t dry out a soap bubble. It’s not stable in mild acid. In general, it must remain in body fluid, so you will transmit this through a respitory way, blood also. HIV is an enveloped virus and it’s transmitted through secretions and blood.
Need to know the difference between these two, because that’s going to tell you whether or not you’re going to get rid of it by just wiping down your counter with a mild detergent. Not going to get rid of the naked capsid, but will get rid of the enveloped.

Icosahedral Capsid Assembly [S11]
The capsid assembly for these icosahedral viruses is a very interesting processes. It’s basically a structural assembly process where small proteins assemble into pentamers. These self assemble into larger structures. Eventually leading to the capsid.
People have visualized this using electron microscopy as well as crystallization and atomic resolution.
If you want a way to visualize this think about trying to put together a soccer ball. They all come out looking like soccer balls.

Examples of Icosahedral capsids [S12]
Here are some examples of some icosahedral capsids. The larger ones are by chriorein (?) reconstitution.
The smaller ones are at atomic resolution, where you can crystallize the whole virus. Depending on how you look at it they mostly look like soccer balls.
What is sticking out of these are actually proteins that the immune system will recognize and bind to that will help neutralize these viruses.

General Enveloped virus structure [S13]
Again, here are two of the viruses that you should be aware of.
HIV is an enveloped virus.
Influenza is also an envelope virus.
HIV has an internal capsid structure that is a little more regular than the nuclear protein RNA structure of influenza.

Example Envelope glycoprotein: Influenza Hemagglutinin [S14]
Flu has two proteins embedded in the envelope—on the outer surface of the virus. One is hemagglutinin. The other is neuraminidase. Hemagglutin binds to salic acid, and neuraminidase clips salic acid and it’s a combination of those two that allow the virus to get in and out of cells. Why is that important? Hemagglutinin is Hand Neuraminidase is N so when you have H and N you have H1N1. These are where the antigentic determinants are and this will determine whether or not you’ll be immune to the particular flu strain or not be immune to the flu strain. These are targeted to one part of the glycoprotein that’s on the envelope.

Steps of virus replication cycle [S15]
This is an important slide. Very important for you to know and remember the steps in replication. He always asks a question about the steps in replication. Important to know the sequence in these steps.
Start out with recognition.
Attachment
Virus has to attack to the surface of the cell. Penetrates the cell. Then it goes through a process of uncoding. The virus is surrounded by either nucleic capsid or viral proteins, they need to uncoat and expose the nucleic acids. Then depending upon the virus (pointing at middle of pathway) the virus goes through transcription-- that means it makes more of its viral genome. Remember that when a virus infects a cell it’s not thousands of things coming into the cell, it’s maybe 5 or 10 at most. Its main job is to amplify itself. Once it does that it makes viral proteins. These then further replicate the viral genome.
Cyle (in middle) continues to rotate and more and more viral genomes. At some point these genomes are encapsidated either:
put into the naked capsid, where the cell is generally exploded by lysis and released.
Or in these envelopes where it goes to the plasma membrane and buds from plasmid membrane. Individual viruses have different pathways.
What you need to know is what’s numbered. Be sure to know the sequence. If you can remember that it will help you with the other viruses that you learn about.
Lytic virus (bottom right corner). We define lytic by the cell lysis. The cell explodes and dies.

Lytic virus growth curve [S16]
When a virus infects it goes through a phase called eclipse. That means that it’s kind of hiding inside the cell. So the virus infects and then the very few virus particles get into the cell. It’s replicating its viral genome. If you break open the cell and try to find infectious virus, you’ll find very little infectious virus. A lot of viral nucleic acids, but little infectious virus. As it goes along, you start to get increased amounts of infectious virus finally to a point where the cells will either burst or lyse. This difference in what you started with and what you end with is called the burst size- that’s the amount of virus that comes out of an infected cell. Viruses have different burst sizes. Some viruses you get tremendous amounts of virus particles outside the cell. Other viruses, like retroviruses, produce much less virus coming out of the cell. The burst size is an indicator of the infectivity of the virus particle. So if you have a high number of virus particles coming in to the cell in order to infect another cell you generally need a lot of particles. Ifyou have a low number of virus particles coming out of the cell, you don’t need as many particles to infect. From the virus point of view, if you’re very inefficient during replication you want to make a lot of viruses so that when you go to infect another cell you going to have a good chance of making a new infection. If you get below the number of particles that you need to infect the cell you won’t infect the cell. The cell will be able to compensate and kill you off.
What you want to avoid viruses that have a high number of particles released but only need a few to infect the cell. Those are very lethal viruses. These are usually exotic viruses. These are need a few particles to infect they cell even though they make a lot of virus. (I’m not sure if he meant to say that they make few particles, or if these exotic viruses are different). They are very pathogenic.

Recognition of cells and attachment [S17]
Viruses need to get inside the cell. They have to have a way to attach to the cell. Viruses just don’t float along in the environment and bump up against cells. They are targeting cells. They have specific attachment sites. So for HIV there is a molecule called CD4 and second molecule called a chemokine. CD4 is found on a subset of T-cells in your body that HIV likes to infect. So when HIV infected patients come down the AIDs all their CD4 cells are destroyed because HIV has targeted all their CD4 cells. If the cell doesn’t have CD4 on it’s very unlikely that it will be infect by HIV. It also needs the chemokine receptors. So it needs this kind of lock and key to infect the cell. It defines the tropism of these viruses.
On the other hand, flu binds to sialic acid. Which is present on a lot of your cells. So flu will infect a lot of different cells depending on the amount of sialic acid. Where are most of the sialic containing cells? –in your pharyngeum and your lungs. It binds to cells with sialic acid, infects those cells, then kills them. The attachment molecules on these cells is very important because as therapeutics have evolved once we know what the virus needs to get into the cell we can design molecules to prevent the virus from getting in. There are peptides that mimic some of CD4/ CCR5, that will bind HIV and prevent the virus from getting into the cells. The one caveat is that HIV can mutate away from this, and move away from binding the peptide and actually binding the cell.

Steps of Replication cycle [S18]
To go back to the picture again, for virus entry, naked viruses (one’s without the envelope) enter the cell through endocytosis.