FUN2: 11:00-12:00
Scribe: Joan-Marie Manolakis
Monday, November 17, 2008
Proof: Caitlin Cox
Dr. Dokland
Immunology
Page 1 of 5
Specific Description of the Topic Being Discussed Today
Introduction [S1]: DNA Viruses
Objective-this is an overview of those viruses associated with DNA genomes. He will emphasize the important human
pathogens. Understand the challenges faced by DNA viruses and the advantages of having a DNA genome (how they
have found solutions to the problems they face). The last point is minor- you don’t have to worry about.
I. This is the list of the families of DNA viruses. [S2]
a. They are grouped into three groups: the main ones are the double stranded DNA genome, a second with single
stranded DNA genomes, and a third group with a unique virus that has a hybrid RNA/DNA genome and
replicate a lot like retroviruses (Hepatitis B virus)
b. Red=important pathogens
c. Most of them do not include human viruses. There are a few that infect bacteria, bacteriophages, and mammals
but not typically humans. He’ll focus on the red families.
II. Sizes of DNA viruses. [S3]
a. They come in a wide range of size.
b. The circoviruses are the smallest and simplest DNA virus. It has a small, single-stranded genome of about
1700 bases. It only codes for two genes. It has a small isometric capsule that is 17 nm.
c. Mimivirus is the largest known virus and has 1.2 Mbp double stranded genome DNA base pairs encoding for
over 900 genes.
d. Mycoplasma has a genome that is half the size of the virus and encodes for about half as many genes. The
Mycoplasma cells are a few hundred nanometers compared to the 600 nm of Mimivirus. This is a virus that
rivals cells in sisal complexity. Like all viruses, it’s dependent on the host cell for its replication. It particularly
infects amoeba.
e. Adenovirus, in the middle, has a capsid diameter of about 90-100 nm, is double stranded and codes for 30-40
genes. It is average for DNA viruses. Compared to RNA viruses, DNA viruses are typically larger and more
complex. DNA replication is more accurate than RNA replication. Thus they can grow larger, more stable
genomes.
f. DNA virus life cycles [S4]
i. There are various strategies and families for replication. There are few on plants.
ii. They range in sizes, i.e. envelope and non-enveloped forms.
1. Some are icosahedrally symmetric, helical cylindrical bullet shaped formed, or more pleiomorphic
(have no defined shape).
2. Some are complex (have components of different symmetries).
iii. The general things about DNA virus: they replicate their genome in a similar way as the host cell. Thus,
they can use the replication and transcription machinery of the host cell. They don’t necessarily need to
encode for their own polymerase. DNA replication is very accurate so they can have very large genomes.
iv. The picture shows a life cycle for a general DNA virus.
1. First, it enters the cells via endocytosis or fusion with the cell (enveloped viruses). Most DNA viruses
replicate in the nucleus because that’s where the replication/transcription machinery of the host is.
This involves uncoating.
2. Once in the nucleus, the DNA genome may be transcribed. mRNA is taken to the cytoplasm where
translation occurs. The proteins are sent back to the nucleus for transcription, replication, and
assembly (all of these occur in the nucleus).
3. They must leave, so some lyse the cell. Sometimes there is a complex transport mechanism.
4. The poxvirus actually replicates in the cytoplasm, so these viruses must bring all the enzymes
necessary for transcription into the cell.
g. Specific challenges for DNA virus [S5]
i. They don’t need DNA/RNA polymerase like RNA viruses. They might encode their own DNA polymerase.
ii. They replicate only during the S phase because it’s the only time that the enzymes for replication are
available in the body. Most are frozen in G1 or another phase, so it is not replicating. In this case, the
virus may not replicate either. They must actively promote cell growth by switching the cell to S phase.
That may lead to transformation of the cell and a cancerous phenotype.
iii. Other viruses use some or many of their own enzymes for replication.
iv. Latency includes viruses that can’t undergo productive growth and it integrates into the host genome where
it can stay for a long time (until it’s activated). Then virus growth will continue.
v. In most cases, they must enter the nucleus intact or disassembled/uncoat. They must also exit the
nucleus.
III. DNA Replication [S6]
FUN2: 11:00-12:00
Scribe: Joan-Marie Manolakis
Monday, November 17, 2008
Proof: Caitlin Cox
Dr. Dokland
Immunology
Page 2 of 5
a. You need a DNA template, DNA polymerase, and a primer. The main DNA polymerases can’t initiate replication
from scratch, rather they need a primer. Typically for replication the primer is RNA that binds to the DNA
template and is a starting point for replication. Most of these are supplied by the host, not the virus.
b. This shows the replication fork of double stranded DNA. It is unidirectional, going from 5’ to 3’. On the leading
strand, as the fork opens you can replicate the DNA in a continuous manner. On the other strand you replicate
in small pieces, each one primed by a small piece of RNA that is subsequently removed (lagging strand).
c. If you have a linear genome and replicate the genome, you will get to the end (primer). It is degraded and
you’re left with a staggered end. Unless there is a way to deal with it, the DNA will be shorter and shorter for
each cycle. Telomerase fixes the ends from getting shorter and shorter. They can get shorter and shorter until
they are no longer viable.
d. Viruses are either circular or there is a protein attached to the end of the genome which serves as a primer.
Then you don’t have problems with the ends.
IV. Getting access to the cellular DNA replication machinery [S7]
a. To get access to the machinery, you must get into the nucleus. Eukaryotic nuclei have nuclear pores that
serve as a means of entry. Viruses enter the nucleus by this method.
b. Some viruses can get through the pore, but some have their genome transported. Others dock on the nucleus
and inject the DNA into the nucleus.
c. Some DNA viruses replicate in the cytoplasm, so they don’t have this problem.
V. Small, ssDNA viruses: Parvoviridae [S8]
a. These are among the smallest DNA viruses. They have about 5,000 nucleotides and are linear, ssDNA. They
code for about 3 proteins.
b. It is a unique kind of DNA. It’s mostly single-stranded but at the end it has terminal hairpins that loop back on
them. Now you have self-priming DNA. When replication starts, it’ll use this primer (thus don’t require a RNA
primer for replication).
c. They are 20 nanometers in diameters. Various parvoviruses infect many animals. FPV is an important
pathogen on cats, and most of them get vaccinated against that virus.
d. B19 virus, Erythrovirus, is the most important disease in humans and causes “fifth disease.” Other childhood
diseases include measles and rubella. They are childhood diseases that cause a red rash. It isn’t very severe
in kids but may be in adults.
VI. Erythem infectiosum (fifth disease) [S9]
a. It’s also known as “slap cheek” disease. It causes a red rash on the cheek and torso (like the child was
slapped).
b. The rash is an immune reaction, so once the virus appears, the rash has cleared. Thus, it isn’t contagious.
i. If a naïve adult is infected with a B19 virus, it may lead to a severe disease in the joints. It’s caused by the
immune reaction because adults have a more robust reaction than children. That’s why many childhooddiseases are more severe in adults.
ii. B 19 is very simple, and it affects only actively replicating viruses. It affects, in this case, those cells in the
bone marrow. There is no vaccine against this one.
c. Parvovirus life cycle [S10]
i. It enters via endocytosis.
ii. It enters the nucleus intact. Replication happens here.
iii. The capsids are assembled in the nucleus, and they get out by lysing the cell.
VII. Parvovirus replication [S11]
a. Linear DNA forms hairpins at the ends. It uses the hairpin as a primer.
b. It unfolds the hairpins, produces a nick, and replicates the rest until the end. Then you must resew (inaudible)
the structure and split it open.
c. There is no need for RNA primer. This is done by the host’s enzymes except the nicking reaction- that’s done
by one of the two genes of the virus itself.
VIII. Papovaviruses: Polyoma- and Papillomaviridae [S12]
a. Polyoma- and papilloma- viruses are very similar. They both have capsids of similar size and organization.
b. Papillomaviruses are larger and more complex.
c. Both have circular dsDNA genomes.
d. The Polyomavirus calls for 5 proteins. VP1 is the main capsid proteins. VP2 and 3 are minor.
e. The other proteins are called large T antigen and small T antigen. They are involved in regulation of
transcription. The large T antigen can turn the cell into S phase and make it replicate. It does so by repressing
one of the tumor-repressor genes. It may lead to a transformed phenotype.
f. Polyomavirus: there are two that infect humans. Papillomaviruses are the cause of warts and cervical cancer.
This is what is being vaccinated against. The L1 is the main protein involved in transcriptional regulation.
IX. Polyomavirus replication [S13]
FUN2: 11:00-12:00
Scribe: Joan-Marie Manolakis
Monday, November 17, 2008
Proof: Caitlin Cox
Dr. Dokland
Immunology
Page 3 of 5
a. It uses host enzymes and proceeds in two directions using leading and lagging strand synthesis and RNA
primer. T antigen is necessary to recruit the genome to the origin of replication-without it, you won’t get
replication. The fate of the virus depends on the level of the T antigen.
b. T antigen:
i. Recruits DNA polymerase to the origins
ii. Switches the cell into S phase so that it’ll start replicating
c. The fate of the virus depends on the host cell
X. Life cycle of polyomavirus [S14]
a. It is necessary to switch the cell into S phase.
b. If it goes into a permissive cell (has a high-enough expression of T antigen), it’ll enter into productive infection
and eventually lyse the cell with the formation of progeny virus.
c. It can infect a cell that’s nonpermissive (has a low T antigen level), but it won’t be able to replicate its DNA
because it can’t recruit DNA polymerase to the genome.
d. It may instead integrate itself into the host genome which may lead to a transformed phenotype.
e. The virus is integrated in the host’s genome, expressing the large T antigen, and that may lead to
immortalization of the cell and lead to cancer.
XI. Papillomavirus infection [S15]
a. Infection enters into the skin and replicates at the right cell type. Here it is in the skin.
b. It leads to warts on the skin. The virus that causes warts is self-limiting and eventually goes away. The most
important thing is that there are certain strains (high risk strains) that infect cervical epithelium, and they may
integrate there and express E6 and E7 oncogenes, leading to cervical cancer. This virus commonly causes
cancer in humans.
XII. Adenoviruses [S16]
a. These are more complex.
b. They have a naked (non-enveloped) icosahedral capsid. They are about 90 nm in diameter with a genome
close to 40,000 base pairs coding for 30 or 40 genes.
c. Several are structural genes and others are involved in replication. It has fibers that are involved to binding to
the host cell for the infection.
d. They have a linear double stranded genome. It could lead to problems in replication of the shortening of the end
pieces. Instead these have terminal proteins bound to the 5’ end of the genomes. It is used as a primer for the
synthesis.
e. They replicate in the nucleus and encode their own DNA dependent DNA polymerase. In part it is due to the
unique priming mechanism with the terminal proteins.
XIII. Adenovirus disease [S17]
a. They are very common. There are many strains and are very involved in an array of diseases. Up to 10% of
respiratory disease in children is caused by these. The typical symptoms include fever, aches, and other
symptoms of cold and flu. It is unlike the common cold virus in that it’s associated with a fever.
b. It is not seasonal- it is spread all of the time.
c. Acute respiratory disease is common in military recruits. It spreads rather easily and is a naked, non-enveloped
virus. It persists and spreads rapidly in a close population. It is a problem in the military, so they used to
vaccinate against it. When they stopped doing so in 1996, they saw an increase in infection immediately.
d. An important disease caused by adenovirus is conjunctivitis and gastroenteritis.
e. It is very resistant and can be transmitted in many ways. It may persist in the body for a long time also.
XIV.
Adenoviral conjunctivitis [S18]
a. This picture is typical. It is a common cause of viral conjunctivitis.
XV. Slide 19: skipped
XVI.
Adenoviruses use a 5’ terminal protein to prime DNA replication [S20]
XVII. This shows the replication scheme for adenovirus. You have DNA with the 5’ protein (the yellow thing) at the 5’
end, so during the replication it’ll split open the dsDNA.
a. Replication will start and proceed from one end to another (not discontinuous at all).
b. The naked strand codes single-stranded DNA binding protein until you can start another round of transcription
from the other end. It’s always 5’ to 3’ starting with the terminal end.
c. No DNA/RNA primers are required, and there’s no lagging strand synthesis
XVIII. Herpesviruses [S21]
a. They are relatively large with dsDNA genomes.
b. 100-300 base pairs
c. They are circular (like the prokaryotic chromosome).
d. They are enveloped up to 300 nm in diameter and have an icosahedral nucleocapsid core.
FUN2: 11:00-12:00
Scribe: Joan-Marie Manolakis
Monday, November 17, 2008
Proof: Caitlin Cox
Dr. Dokland
Immunology
Page 4 of 5
i. The core has three components. It has an icosahedrally ordered core, a tegument layer, and the envelope
itself. There are various proteins within this virus.
ii. Herpes virus contains many important human pathogens.
XIX.
Herpesvirus diseases [S22]
a. Herpes simplex virus 1 and 2 are fairly ubiquitous.
b. Varicella is the positive agent of chicken pox in kids/shingles in adults. Part of this is due to the latency that
viruses undergo. They’ll infect at certain sites, particularly epithelial cells for this virus. Then they’ll move
somewhere else (where they’re latent until reactivated).
c. Others viruses are involved in birth defects and other pathology. Epstein-bar virus is a causative agent of
mononucleosis.
XX. Herpesvirus life cycle
a. They have a complex life cycle compared to others. It is divided into three stages.
i. The first stage: the virus enters into the cell. This one is enveloped. It fuses with the plasma membranes,
releasing the nucleocapsid into the core (this word wasn’t clear in audio). The DNA needs to get into the
nucleus but the nucleocapsid is too large to be transported into the nuclear capsid. It will dock at the
nuclear pore and inject the DNA into the nucleus.
ii. Once the DNA is inside the nucleus, the immediate early transcription occurs. As it transcribes a small
subset of proteins (the immediate early genes) which are involved in transcription regulation. The
transcripts need to get to the cytoplasm, and then they are reimported into the nucleus. These proteins
bind the viral and host DNA and stimulate transcription of certain genes. The second set of genes
transcribed is early transcripts.
iii. Now it’ll start producing mRNA and proteins involved in DNA replication of the virus. It sets up a nuclear
factory where the transcription and replication will occur. It’ll produce proteins involved in these processes
and starts replicating the genome.
iv. The third and “late transcription” phase is when more of the virus is produced. You start producing the
structural proteins that are needed to make the nuclear capsid integument and the envelope structures. It’ll
now set up a cytoplasmic factory (assembly compartment). The nucleocapsids are produced in the
nucleus and exit by a process that isn’t well understood. They undergo many steps of integumentation (the
addition of the outer layer), and eventually bud into cellular compartments (where they acquire the
gylcoproteins for the envelope). Eventually they’re released out of the cell; it many occur by exoctyosis
(cell isn’t lysed) or lysis. In other cases it may causes cells to fuse together and make a huge large cell,
spreading in that way.
v. The mechanism is known as a “rolling circle mechanism.” It’ll nick the DNA, replication occurs in a
discontinuous manner, and as it unrolls it spins around and churns out a longer piece of DNA. Eventually
it’ll be multiple genomes together. It’s then packaged into the preformed nuclear capsid using an enzyme
called terminase. It packages the DNA and caps it.
vi. Viral latency: a hallmark of herpesviruses. You may get infection in primary sites, but when it gets
to secondary cells it may enter into neurons. It’s transported into the nucleus of the neuron. It
doesn’t replicate there, but it may stay there for a long time as plasmid DNA. Some process may
reactivate the virus.
vii. Example: he talks about a child that had the chicken pox virus which later manifested as shingles (at an
older age). They may be activated by stress or other factors.
XXI.
Cytoplasmic DNA viruses: the exception to the rule [S24]
a. These viruses replicate in the cytoplasm. The most noticeable is the smallpox virus.
b. They need to synthesize all of the enzymes involved in replication since that’s not available in the cytoplasm.
Thus they are very large and complex. They code for a lot of proteins (polymerases, helicases, etc.).
Everything you need is encoded by the virus.
c. Replication takes place in a virus factory.
d. Cow pox forms the basis of the vaccine for smallpox virus. [S25, 26, 27]
e. Orf (sheep and goat pox)-can infect humans but normally don’t lead to serious disease. However, they don’t
spread. They stay as localized lesions. They’re normally seen on animal handlers and veterinarians.
i. The genome is partially double stranded DNA.
f. Poxviridae Phylogeny [S26]
g. Monkey pox may infect humans. It can spread throughout the body. Various forms may lead to small pox.
XXII. Replication cycle of vaccinia virus [S28]
XXIII. This is the life cycle of smallpox. Everything occurs in a region of the cytoplasm. Assembly occurs through
various steps, leading to a virion with several layers. In the cell it forms the replication complex where replication
occurs in the early phases. Eventually it gets to a viral factory (immature viruses that undergo maturation).
XXIV. DNA viruses: Things to consider [S29]
FUN2: 11:00-12:00
Scribe: Joan-Marie Manolakis
Monday, November 17, 2008
Proof: Caitlin Cox
Dr. Dokland
Immunology
Page 5 of 5
XXV. Think about DNA viruses in terms of what challenges does it face and what strategies does it employ to resolve
these challenges?
a. How does it get into the cell? How does it get into the nucleus? Is it intact? Is it disassembled? Where and
how does it replicate its DNA? What does it need to replicate itself? What can it use that the cell supplies and
what does it need to bring? How does it deal with the fact that cells only replicate themselves in S phase?
Latency? Activation?
b. This is how he’d think about these viruses.