Microbiology
10/13/2008
Introduction to Virology—Dr. Morrow
Transcriber: Haleigh Stidham
51:29
1) Introduction to Virology. You will only be responsible for what is on this lecture!! There will be
no trivial details. We are going to talk about viral replication—how viruses exploit cells to make
new ones, and viral pathogenesis—how viruses cause disease and escape from the immune
system.
2) One of the things you want to remember when we talk about viruses is the molecular biology
dogma, and that dogma is DNA -> RNA -> Protein. DNA in eukaryotic cells is in the nucleus; it
gets transcribed to make mRNA; transported into the cytoplasm, and translated into proteins.
Viruses exploit this pathway in your cells.
3) So what are the general characteristics of viruses? They are “filterable agents.” They pass
through filters that catch bacteria. They are obligate intracellular parasites—a really important
concept. They need cells in order replicate. They need the host’s translational, DNA, and RNA
machinery. They have minimal genetic information and don’t have large amounts of DNA.
Although some such as the Herpes virus are large. They rely on host cells machinery to encode
DNA. They are assembled from building blocks encoded by the virus. Viruses absolutely require
a host cell for replication to produce proteins to synthesize new viral genomes and the building
blocks of virus structure.
4) For a virus to be successful, it has to be capable of transmission through potentially harsh
environmental conditions. The pathway of transmission is mostly fecal-oral. This means that
they are stable in the gut and stable in the environment. A lot of viruses are transmitted on
aerosol droplets (ex: in an elevator). The skin is a fantastic barrier but the mucosal surfaces are
not that great. Viruses need to get through the skin and other barriers from the host. The
mucosal surfaces (ex: mouth and eyes) are not that great to keep viruses out and that’s how
they are transmitted. They must adapt to the biochemical machinery of the host cell for
replication. Most host cells are not actively replicating, and the virus has ways to get the host
cells to replicate so that they can replicate. They must escape elimination by the host immune
response.
5) The basic components of a virus or virion are that they are composed of DNA or RNA. They have
structural proteins that surround this RNA or DNA because they have to be protected from the
environment. Some viruses have enzymes that are encapsulated into the virion. Those enzymes
are virally encoded enzymes that help viruses infect cells. Nucleic acids are surrounded by a
nucleocapsid protein to help protect them from the environment. A nucleocapsid can also be
around DNA or RNA and surrounded by a host cells membrane, or an envelope. The best way to
think about this is a “soap bubble.”
6) Viruses have been around a long time and there are many ways to classify them. They are
classified by size, morphology, the way they look through an electron microscope, genome type
(RNA or DNA), and their needs of replication. DNA viruses are classified into envelope viruses
(surrounded by a lipid membrane), and those include: Pox virus (small pox), Herpes virus, and
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Hepadna virus (liver viruses). If they have a naked capsid, which is usually icosahedral, it
includes: Polyoma, Papilloma, Adeno, and Parvoviruses. If the nucleic acid is RNA, there is a lot
of diversity. There are Plus stranded RNA—Picornaviruses (Cold and Polio viruses) and
Togaviruses. The Negative stranded RNA viruses include: Rhabdo (causes Rabies), and
Orthomyxo and Paramyxoviruses (causes the Flu). +/- Double strand RNA viruses are
Retroviruses. They start out as RNA and use reverse transcription to DNA (HIV).
7) The sizes of viruses. A tube of E. coli. One of the largest viruses is the pox virus, which causes
small pox. You can see these with a light microscope. But for the most part, you have to use an
electron microscope to see these viruses. The smallest is the Picornavirus, which causes Polio
and Rhinovirus. The Rhabdovirus causes Rabies and is shaped like a bullet.
8) The general structure of viruses can be looked at in two ways. One is the naked capsid virus—an
icosahedral structure that surrounds a nucleic acid. A second structure of virus is the
nucleocapsid. It is actually a small protein that binds to the viral nucleic acid, usually the RNA.
This is position inside of an enveloped virus. Many times it is not a sphere and can have many
shapes, unlike the structured icosahedral virus.
9) The viruses are mainly divided by genome, DNA and RNA content. In general, RNA viruses have
a wider array of sizes than DNA. Also, DNA viruses are larger in size than RNA.
10) A naked capsid is not surrounded by a lipid. It’s almost entirely proteins that surround a
nucleic capsid. They are resistant to drying, acids, detergents, and are spread in the fecal-oral
route. You cannot kill them with Lysol. On the other hand, Lysol can kill enveloped viruses.
Lysol could kill a tube of ‘flu’ (enveloped) but not a tube of the ‘common cold, hepatitis A, or
polio virus’ (naked). You can kill naked capsids with an autoclave.
11) Icosahedral assemble from small protein building blocks into larger pentamer and then into the
larger icosahedral structure, like “soccer balls.”
12) Examples of icosahedral capsids are determined by x-ray crystallography.
13) Enveloped viruses include HIV and Influenza. HIV has an envelope surrounding the viral capsid
and forms a bullet-shaped structure. The nucleocapsid surrounds the viral genome and
surrounded by an envelope. Embedded in this envelope is the gp120 that binds to the CD4.
This is a flu RNA virus. It contains eight segments of RNA surrounded by a nucleocapsid and this
lipid membrane is embedded with hemagglutinin and neuraminidase.
14) This is an example of the envelope glycoprotein, the Influenza hemagglutinin. This is the protein
that is embedded in the membrane and what the virus uses to interact with cells.
15) ****This is an important slide!!!**** You need to remember the certain steps that a virus must
go through to replicate. This will help you to distinguish between all the viruses we are going to
talk about. The virus is floating around out here and it needs to interact with the cell in a
process called 1) recognition and 2) attachment. For example, HIV binds to cells that express
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CD4 on T cells and not B cells because they don’t have CD4. On the other hand, the Flu attaches
sialic acid, which is on a lot of different cells. So it will attach and infect a lot of different cells.
After this, it goes through the process of 3) penetration and 4) uncoating. Basically what
happens is the virus gets enveloped into a vesicle. The virus knows this and will get out of the
vesicle into the cytoplasm. The virus is uncoated and releases its nucleic acids into the cell. If it
is a DNA virus, it’s going to go to the nucleus to get its viral genes expressed through 5)
transcription. The virus will then continue with the dogma to RNA and then 6) protein synthesis.
If it is an RNA virus, many times it does not have to go to the nucleus, it can just start in the
cytoplasm. So this will change depending on the virus. ****“I guarantee you that I will ask you
a question about this when we talk about Picornaviruses because they have a unique way of
replication.”*** So you need to know these first steps, and steps 5, 6, and 7, will change
depending on the type virus. After 7) replication, the last step of the virus is to 8a) assemble
and actually get outside of the cell. One way to get out is that the cell replicates so much that it
explodes, it 9a) lyses. That is what we call a ‘lytic’ virus. Other viruses actually will go to the
plasma membrane for 9b) envelopement and through a process of 9b) ‘budding’ will release
new viruses into the environment. These are enveloped viruses that get it from the host
membrane. Generally, budding viruses are not lytic on the cell.
16) Here are some growth curves of some lytic viruses. Picorna, Rhabdo, and Togaviruses put a
large yield of virus per cell in a very short time. Other viruses such as the Retrovirus bud from
the membrane and produce a small amount over time. It does not cause lysis of the cell.
17) In order for HIV to infect the cell, the glycoprotein embedded in the membrane, gp160 (made
up of gp120 and gp41), must attach to CD4 T cells by gp120 and the chemokines CXCR4 and
CCR5. In contrast, Flu binds to sialic acid present on a lot of different cells. Once it binds the
sialic acid, the membrane fuse and it dumps its nucleic acids into the host cell.
18) Naked viruses do not have a lipid membrane. They use endocytosis for cell entry. They can
withstand the harsh environments of the lysosomal vesicles. They can pop these lysosomal
bubbles and release their nucleic acids into the cytoplasm. Enveloped viruses use cell fusion
with the membrane and release their nucleic acids into the cytoplasm.
19) Once the virus gets into the cytoplasm, it wants to release and synthesize its nucleic acids. It will
make mRNA encoding viral proteins and new viral genomes for encapsidation. This happens
early in the infection. Later in the infection, viral proteins will be enzymes and other proteins
required for viral transcription and genome replication. There are also structural proteins. The
enzymes are targets for antivirals.
20) All of these take advantage of the cellular process of DNA to RNA to proteins. Viruses have a
unique problem when they infect the cell. They must out compete the host to get their mRNAs
translated.
21) DNA virus transcription uses the host’s DNA-dependent RNA polymerase to make mRNA. The
RNA polymerase II is the host cell’s enzyme that is used to make the mRNA. DNA genomes go to
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the nucleus and integrate into the host’s chromosomal DNA. Once integrated, you can’t get rid
of it. The only way to get rid of it is for the cell to die. This is why you can’t treat HIV patients.
There is one exception, the Poxvirus. The Poxvirus is a large DNA virus that replicates in the
cytoplasm of the cell and does not use the hosts RNA Polymerase II. It makes its own enzymes
for transcription. This is important because if you want to make antiviral drugs for small pox,
these enzymes are excellent targets.
22) RNA transcription. Many RNA viruses replicate entirely in the cytoplasm. They need an enzyme,
RNA-dependent RNA polymerase. ***This is another point that you need to remember—how
do we name these polymerases???*** The name tells you how it works. It’s ‘RNA dependent,’
meaning it needs RNA and it’s an ‘RNA polymerase,’ meaning it makes more RNA. The host cell
has not enzymes for generating a RNA template, so this is very good target for RNA antivirals.
****This is another point that you need to remember—and that is how we define RNA
viruses!!!**** Plus (+) strand RNA viruses—genomes are the same sense as the messenger RNA. So if
you take a (+) strand RNA and put it into the cytoplasm of a cell, it can be directly translated to make
viral proteins. Minus (-) strand RNA—genomes are in the opposite sense as mRNA and is the
compliment of a (+) strand. If you put a (-) RNA into the cytoplasm of a cell, it’s dead. It cannot
translate and cannot replicate. So remember that this will change if you have a (+) strand and a (-)
strand. ***SLIDE 15—remember steps 4, 5, and 6 (I think he meant steps 5, 6, and 7) will change
depending on which strand you have during replication.
23) Replication of viral genomes. DNA viruses use a DNA-dependent DNA polymerase. So they
need DNA and they make more DNA. Some use cellular DNA polymerase, except the Herpes
virus which makes its own DNA polymerase. This is a good target for antivirals. RNA viruses use
RNA-dependent RNA polymerase. It is encoded by the viruses. Retroviruses (ex: HIV) genome
is a single-strand RNA. HIV packages an enzyme in its virion called Reverse Transcriptase.
Reverse transcriptase is used to form DNA from RNA. This DNA then integrates into the
chromosomal DNA. The new viral genomes are now transcribed in the nucleus from the
integrated DNA. It is in the DNA forever. The antivirals for HIV are targeted at this Reverse
Transcriptase (RT) because this enzyme is not found in normal cells. But the antiviral drugs can
never stop all the RT.
24) Viral protein synthesis. Generally, they all use the host cell’s translational machinery. Many use
a process called polyprotein strategy. There are multiple proteins encoded on one mRNA. The
individual proteins are derived from polyprotein by enzymatic cleavages by proteases.
25) Cartoon of a Picornavirus. This virus is translated as a large protein. This little protein here
called 3Cpro will go around and make cleavages at specific amino acid pairs to make proteins.
For example, 3CD contains the vital protease and has proteolytic activity on the capsid proteins.
It contains 3Dpol which is the RNA polymerase but has no protease activity. By regulating the
processing, the virus can make different functional proteins from the same polyproteins.
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26) We have talked about assembly and release. Enveloped viruses exit by budding from the cell.
Naked viruses exit through cell lysis. They explode the cell with so much virus in the cytoplasm.
27) Viral budding is a very interesting process. These particles are released from the cell. The cell is
fine after the virus leaves, but it is producing lots of viral particles.
28) Viral pathogenesis is the interaction between the virus and the host. The two general routes of
viral infection are fecal-oral and inhalation. Viruses have no incentive to kill you, so they stay at
the primary site of replication. They want you to produce more viruses and if you are dead, you
can’t produce any virus. Some viruses have a viremic stage, where they leave the primary site.
This secondary site of replication is usually what causes disease.
29) One example is Ebola. This virus infects macrophages and migrates to various organs. You get
viremia from going to the secondary targets. This is where the severe problems occur.
Characteristically, you bleed out from the inside with the Ebola virus.
30) Outcomes of the virus infection. Usually, you have a failed infection. The host cell wins and the
virus is aborted. You can have cell apoptosis when it realizes it is infected. You can also have
infection without cell death, which this can cause chronic disease. A latent infection such as the
Herpes virus, are viruses that hide from your immune system and come out during periods of
stress or sunburns. Some viruses will take the cell that they infected and transform it. This is
what Retroviruses do. They integrate into your chromosome and cause transformation, which
can lead to cancer.
31) We have evolved ways to protect against viruses. The best barrier against viruses is your skin.
Viruses can get into broken skin and mucosal surfaces. We also have an innate immune defense
that is not dependent on antigens. There is an interferon response. Macrophages gobble up
viruses and digest them. Dendritic cells do the same thing. Natural killer cells kill anything in
your body like virally infected cells or cancer cells. Also, we have an antigen-specific immune
response. If you get vaccinated, we make antibodies against that particular virus. If you come
in contact, the antibodies bind to that virus to get rid of it. Helper T-cells, CD4, help with
recognition of virally infected cells. Cytotoxic T cells lyse the infected cells and they cannot
replicate.
32) The viruses aren’t dumb. They can prevent Interferon infection. Double stranded RNA can
negate Interferon. One process that occurs over and over is the changing of viral antigens. This
is true for the flu virus and HIV. These viruses change their surface antigens and our immune
system can’t keep up. Viruses have evolved to have cell to cell spread. They don’t get out into
the blood and the antibodies can’t get inside of the cell. They also have ways to suppress
antigen presentation and hide from the immune system.
33) Immunopathogenesis. Most of the time when you get a cold as the result of a viral infection,
the symptoms are not from the virus. Your immune response causes the symptoms as it
prevents the virus from replicating. The flu-like symptoms are caused by interferon and
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cytokines. What happens when you get a fever? Your body temperature rises to slow down the
replication of the virus. Inflammation and delayed-type hypersensitivity are other ways. Many
times you seem like you are getting sicker, but it is your body resolving the infection. Some
times immune complexes are formed when an antibody for a virus reacts to a protein in your
body. These can cause immune complex disease and can lodge in your kidneys, activating
complement. Postinfection cytolysis happens with T cells. Immunosuppression happens with
HIV, CMV, and measles. The cells involved in the normal way of keeping your disease free are
killed, and then you become susceptible to all the environmental factors. It’s not HIV causing
disease, it’s mainly HIV killing the CD4 T cells. It’s the secondary effect when you become
infected with disease that you would normally be able to fight off.
4 things he added after the recording stopped:
1. Slide on replication is important SLIDE 15
2. Know the difference in (+) and (-) strands SLIDE 22
3. Know about polymerases SLIDE 22 & 23
4. Know the difference in enveloped and naked virus SLIDE 10, 18 & 26
SOOOOOO….basically these are the four questions he will ask you on the test!!!! 