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Microbiology
10/13/2008
Introduction to DNA viruses
Transcriber: Laura Rayne
49:48
Slide 1: We’re going to continue talking more specifically about DNA viruses. This is going to be a
general overview of DNA viruses.
Slide 2: This is the known family of viruses that have DNA genomes. This taxonomy is continually being
revised and don’t have to memorize families, just gives an idea of the diversity that viruses have. It’s
divided into 3 groups the biggest one being the double stranded DNA (dsDNA) genome, there are
another few families that have a single stranded DNA genome and a third group that’s very unique. They
have a DNA genome but replicate using an RNA intermediate, the most important viruses for our
purpose is Hepatitis B virus. These are more related to retro viruses and they’ll be another lecture on
them later.
The ones highlighted in red are the families that infect humans, as you can see there are some
important pathogens here, like the adenoviruses, herpes viruses, papilloma virus, and pox viruses. Most
of these families don’t infect humans or even mammals but are invertebrate viruses and bacteria. What
doesn’t come across in this scheme is that there are relationships between these groups as well, so
there are evolutionary relationships between viruses that infect humans with those that infect bacteria
suggesting that viruses are very ancient. A few of these families here, such as Asfarviridae and
Circoviridae do infect mammals, but not humans. Since viruses are continuously evolving, it’s not a
stretch to think that human pathogens can arise from those families.
Slide 3: The DNA viruses encompass a very diverse group and if you look at the size and complexity here,
at one end of the scale you have Circo viruses, a porcine pathogen, which are among the smallest and
simplest viruses at 17 nm in diameter, a small icosahedronal capsid and a handful of genes, with a very
small ssDNA genome.
At the other end you have Mimi viruses, which infect amoebas; it’s a huge virus with a million bp
genome, over 900 genes and a size of about 600 nm. Compared to Mycoplasmas, this viruses genome is
twice as big with double the genes, but it’s still needs a host cell to replicate. It starts to blur the
distinction between viruses and other forms of life. On average, DNA viruses tend toward the larger end
of the scale. An adenovirus is a typical example with few tens of genes and genome with 30-50 thousand
bps.
Slide 4: So what really distinguishes DNA viruses from other viruses? The DNA genome. There’s a wide
variety of different life cycles among DNA viruses. Main thing is that since the genome is dsDNA like the
host cell that they infect, they can in principle use the same replication and transcription machinery that
the host uses. This scheme here shows a general life cycle that’ll be true for most but not all DNA
viruses. They get into the cell through some mechanism. There are enveloped and non enveloped forms
and this will determine how they get into the cell.
The DNA genome has to make it into the nucleus in most cases to the replication machinery. Proteins
are synthesized in the cytoplasm, and then are shipped back into the nucleus where most assembly
happens. To exit the cell, they may have to go thru the nuclear envelope into the cytoplasm and then
bud. Or they exit by lysing the host cell.
Microbiology: Introduction to DNA viruses
Laura Rayne
pg. 2
Most replicate and partially assemble in the nucleus, but the exception to that is the pox virus that
actually replicate in the cytoplasm, and we’ll cover that in a minute.
Don’t forget the proteins are made in the cytoplasm so there has to be transport of RNA out of the
nucleus and then proteins back into the nucleus in order to assemble.
Slide 5: Skipped
Slide 6: At least in principle the viruses can utilize the replication and transcription machinery of the
host, which means RNA genomes need a virus encoded polymerase to replicate its RNA, because the
host does not have a function for replicating RNA. Some of these DNA viruses do call for their own DNA
polymerase anyway. Most cells in the body do not replicate most of the time, so in order for the virus to
use the cell’s machinery, it needs to infect cells that are actively replicating. Most cells are frozen in G1
before the S phase. So the virus will convert these cells to an actively replicating state, which means it’ll
convert the cell to a cancerous phenotype that’s actively growing. For this reason, some viruses do
produce their own replication proteins to by pass this requirement for growth.
Another strategy that some DNA viruses use is that they go into a latent state, which is a prolonged
period where there is no production of virus. The virus exists as just an episome or a naked DNA in the
nucleus and can sty there for many years. This is very typical of Herpes virus and can be reactivated.
Another strategy is to integrate into the host genome which can cause a lot of problems. Unlike RNA
viruses that just have to get into the cytoplasm of the host cell, the DNA virus has to get access to the
nucleus, and they need to exit from the nucleus, which may require complex mechanisms for budding
out of the nucleus or will have to lyse the whole cell.
Slide 7: Let’s have a look at DNA replication in general. So what do you need? You need a template
DNA which is the viral genome, a DNA polymerase (supplied by host or virus) but most importantly you
need a primer. DNA replication is always 5’→3’. With a dsDNA that’s being replicated, it’ being opened
and have two strands, the leading strand (continuously replicated) and the lagging strand
(discontinuous) because it has to start from some primer. The primers are in the cell, is a piece of RNA
that’s synthesized and which is then removed later (?).
How do viruses deal with this? Some viruses use the same strategy as the host cell, but there are some
variations on that, and I’ll come back to the reason for that. Some viruses use a protein attached to the
DNA itself as the primer. The problem is if you have a linear dsRNA, how and where you start the
replication. If you start with the primer you’ll end up with a bit of primer at each end and when you
remove the primer, there’s no way of filling in this primer, so you’ll lose a little bit of the genome every
time. For eukaryotic chromosomes, this is a problem considered on of the reasons why cells age,
because you’re chipping away at the end the whole time. Eukaryotes also have telomerase, which adds
in at the end here, this wouldn’t work for viruses so how do you prevent the viral genome from getting
shorter like this. One of the solutions to that is to have a circular genome, which is what the prokaryotes
have. Those that don’t have a circular genome use other mechanisms to prime DNA synthesis including
the use of proteins covalently attached to one end of the genome or hairpins so they can prime their
own synthesis.
Microbiology: Introduction to DNA viruses
Laura Rayne
pg. 3
Slide 8: As I mentioned, you have to get into the nucleus. Cells have mechanisms for getting things in
and out of nucleus that the viruses can make use of. Some viruses are small enough hat they can get
into the nucleus intact, others disassemble in the cytoplasm and have proteins that shuttle the genome
into the nucleus. Others stop at the envelope and inject the DNA like a bacteriophage infects bacteria.
This is how herpes viruses do it, and these are related to bacteriophages in many ways. The final
solution is that some viruses don’t replicate in the nucleus but in the cytoplasm, this requires the virus
itself to bring everything it needs for a replication.
Slide 9: Let’s go through a few virus families specifically. The smallest and simplest viruses that infect
humans are the parvoviruses. They have about 5000 nucleotides and are ssDNA, and has 20 nm
diameter with an icosahedral capsid. There is mainly one that is pathogenic to humans called V19 or
erythrovirus, which is the causative agent of “fifth disease”, which is a generally common and mild
childhood disease.
There are many other parvoviruses that affect animals. You may have heard of the feline form of this
virus or feline distemper virus. You vaccinate cats against this.
There’s also a virus called adeno associated virus in humans, which is a parvo virus that must infect cells
which already infected with adenovirus.
This is the genome. The genome of parvoviruses has a terminal hairpin. It’s single stranded mostly
except at the end that has the hairpin loop that serves as a primer so it can self prime it’s own synthesis.
Slide 10: This is a typical appearance of fifth disease, this erythema infectosium. It’s caused by the V19
virus and is a mild disease in children; it presents with fever, rash, the usual. This rash appears after a
few days to a few weeks. This again is a parvovirus so it must infect actively replicating cells, they don’t
have the ability to convert the cell into a replicating form. They infect erythroid precursor cells in the
bone marrow. The rash is actually an immune response and is not infectious. The virus is already
cleared when the rash appears, unlike chicken pox, where the rash itself is the replicating virus in skin.
There is no vaccine available. It is generally a mild disease in children. When adults get this virus when
not exposed to it as kids, they mount a more robust immune response, and this causes a worse form of
the disease. A typical symptom is arthritis.
Slide 11: So the parvovirus life cycle is a really simple life cycle. The viruses infect the cell by
endocytosis. They enter the nucleus intact because they are small enough to be transported through the
nuclear pores. Replication occurs only if it infects actively replicating cells. How do they get out? They
lyse the cell. It has no mechanism for exportation, so lyses the nuclear and cellular membrane, generally
not a problem for the patient.
Slide 12: Just a few words about the replication. Virus has inverted repeat at the end, which gives rise
to a genome that has these “ears” here. When it replicates it will start here at this 5’ end, replicate the
genome and go all the way to the end by unwinding this hairpin then it’ll nick the other end and will
replicate that to the other end. Now these strands have to be separated, and the hairpins are
regenerated.
Microbiology: Introduction to DNA viruses
Laura Rayne
pg. 4
Slide 13: This is a finer point that is not really important for you. This is the structure of capsid protein.
There are 60 of these, 60 copies of the protein in the virus. This is known as a Beta sandwich or Beta
barrel role.
Slide 14: Papovaviruses are a group that includes two families – Polyoma and Papillomaviridae. These
have many similarities. This is the structure of polyomavirus. It’s “T=7” icosahedron, it’s 72 pentamers of
the capsid protein and has 5000 bp circular dsDNA genome. Polyoma only has 5 genes. It’s pretty simple
and small. The important thing for the purpose of pathogenesis is that they code for 2 proteins called
the T antigens, the large and the small. These are transcriptional activators. They do 2 things. They
switch the cell to activated cells (S phase), and it also recruits the host cell DNA polymerase to the
genome for replication.
Papillomaviruses are much the same; they are slightly larger with a few more genes and are human
pathogens. They normally cause warts but they are also involved in cervical cancer, which is why there
is now a vaccine for it. Both of these have the ability to cause a cancerous phenotype, which is related to
this activation of the cells to go into S phase as well as there ability to integrate into the host genome
permanently.
Slide 16: We have a circular double stranded genome. It is replicated in much the same way as host
DNA using RNA primers. There is a leading strand and a lagging strand. This is known as theta
replication because it looks like the Greek letter theta. So you have 2 replication forks. The T antigen is
required to bring the DNA polymerase to the origin and it replicates and you end up with 2 circles. So
except for the T antigen it only uses host proteins.
Slide 15: So what is important for polyoma and papilloma viruses? It depends on what kind of cell it
infects. This T antigen is required in order to replicate. Whether it is able to switch the cell into S phase
depends on the cell itself. It depends on whether the T antigen expression is high enough in that
particular cell. So there is specificity there as to which viruses can actively replicate. If you are in a
permissive cell type that does allow replication and does allow this switch into S phase, you get a
productive phase and the virus replicates, the cell lyses etc. However, if it goes into a non-permissive
cell, it can’t replicate and may get integrated into the genome. So now when the cell divides, the viral
genome will be replicated along with the host cell. Now you have a permanent expression of this T
antigen and over time it can lead to a cancerous phenotype. So it is a transforming cycle. The cells are
transformed into active growth without the production of virus. There is not enough T antigen to
replicate the genome but there may be enough to allow the cells to start replicating.
Slide 17: Skipped
Slide 18: Adenoviruses are naked viruses. They do not have an envelope, just like the parvoviruses and
polyoma viruses. Adenoviruses are among the largest of the non-enveloped viruses. This is an electron
micrograph of an adenovirus. It has an icosahedral capsid the diameter of about 90nm. Here is a 3D
reconstruction. Although you don’t see it in the micrograph it has these very fine fibers sticking out that
are responsible for binding to the host cells during the infection.
Microbiology: Introduction to DNA viruses
Laura Rayne
pg. 5
Adenoviruses actually have a linear DNA genome. This is one of those where you have a terminal
protein covalently bound to the 5’ end of the genome and that actually serves as the primer for the DNA
synthesis. So the adenovirus also encodes its own DNA polymerase to take advantage of this terminal
protein primed synthesis.
Slide 19: Adenoviruses are very important human pathogens that cause a wide range of diseases. It is a
very common cause of conjunctivitis and is probably the most common cause of viral conjunctivitis. Of
course, there are a number of other things caused by adenoviruses as well.
Slide 20: Adenoviruses do need to get into the nucleus. They replicate in the nucleus like most DNA
viruses. They are non-enveloped. It is not quite clear exactly how they get into the cytoplasm. It is
probably involving endocytosis, but even if it gets endocytosed it still has to somehow cross a
membrane at some point and it is not clear exactly how it does that. It is too big to enter into the
nucleus, so it will bind to the nuclear pore and only a DNA complex will enter through the pore and into
the nucleus.
Slide 21: Here is an overview showing you how this terminal protein-based DNA replication happens.
You will have the terminal protein bound at one end. (Oval=the terminal protein in the picture). It will
start splitting up one of the strands here and replication starts from this terminal protein. A new
terminal protein binds here and it will continue through until you have done the whole thing. So you
end up with a single strand of DNA, which has to be protected. It is protected by a ssDNA binding
protein that is also encoded by the virus. You have the same thing happening on the other side, and this
way you replicate the whole genome without having a problem with the primers at the end.
Slide 22: Skipped. “The structural stuff is not really that important for you.”
Slide 23: Herpes viruses are another very important group of viruses in terms of human health. These
are very large and complex viruses. They have genomes in the range of 100,000 to 200, 000 base pairs.
It is a circular DNA. They code for a large number of proteins, including those involved in transcription
and replication as well as many structural proteins. These are enveloped viruses.
Here is an electron micrograph. They have an icosahedral nucleocapsid core surrounded by an
envelope. It probably doesn’t have this shape normally because this is a negative stain micrograph and
the envelope kind of collapses. It is probably more or less round. There are a large number of proteins
between the nucleocapsid and the envelope, which is known as the tegument. And then the envelope
itself has a large number of envelope glycoproteins, which are involved in the actual infection process.
There are many human pathogens in this group, including herpes simplex virus, cytomegalovirus,
chicken pox, Epstein-Barr etc.
Slide 24: The herpes virus life cycle can be divided into 3 stages. The first one starts upon infection.
This is an enveloped virus. It fuses with the membrane to release the nucleocapsid into the cytoplasm.
This now has to get to the nucleus. Since these are too large to get to the nucleus on their own, they
dock with the membrane and inject the DNA into the nucleus.
Microbiology: Introduction to DNA viruses
Laura Rayne
pg. 6
As soon as it is in there it will begin transcribing a small set of proteins. That is known as the immediate
early phase. The first proteins to be produced are transcription factors involved in initiating
transcription and replication of the genome.
These transcription factors make it back into the nucleus and start expressing a large number of what
are known as early transcripts. Early transcripts are all those proteins involved in DNA replication itself
as well as construction of the nuclear factory, a localized region in the nucleus where replication takes
place.
The start of replication itself is the start of late phase, which is the production of new virus. This is also
when the structural proteins are produced. The structural proteins are proteins that are part of the
virus itself. In this late phase, the DNA is replicated and the virus is assembled. The nucleocapsid is
assembled in the nucleus itself, and this is then transported into the cytoplasm through a rather
complicated mechanism that involves budding into and out of the nuclear envelope. There are a whole
range of additional steps where the virus acquires this tegument layer and then eventually buds into
some internal compartment. This actually takes place in a region called the assembly compartment, a
specialized region of the cytoplasm. When it buds in it acquires its glycoproteins and eventually it is
released into the medium, either by exocytosis or lysis of the cell. These viruses are able to move
directly from one cell to another by forming this large syncytium like cytomegalovirus. It causes all the
cells to fuse together, and this way it avoids the immune system because there is no virus released in
the medium.
New slide - Viral Latency: Herpes viruses are particularly famous for undergoing viral latency. In this
case you may have an initial productive infection, chicken pox for example. You may have replication in
the skin which gives rise to the typical chicken pox blisters, but that infection is quickly cleared by the
immune system. What the virus is able to do though is infect peripheral neurons, travel up the axon
into the nucleus of the neuron where it will stay as an episome, or a naked DNA, without any production
of virus. It may stay there for months or years until by some mechanism, probably immune system
related, it gets reactivated. At that point it will start producing viruses again, which will travel down to
peripheral areas such as the skin and cause a new productive infection. This gets cleared by the immune
system eventually, but the virus is still there. That is why they say herpes is forever. You never get rid of
it. So the varicella zoster virus, which causes chicken pox in children, may cause shingles when it is
reactived in the adult.
Slide 25: The Herpes virus genome is replicated by the rolling-circle mechanism, which is the same way
that bacteriophages are produced. The circle is opened here and it will unwind and produce a long
concatemeric DNA. (“The numbering is not quite right”.) It will produce a long concatemer containing
many genomes that is now packaged into the preformed capsid and cut when the capsid is full.
Slide 26: There are a group of viruses that do not follow the rule that DNA viruses always replicate in
the nucleus. These are cytoplasmic DNA viruses. The important ones for us are the pox viruses,
including smallpox. These viruses actually replicate in the cytoplasm. They don’t get into the nucleus at
Microbiology: Introduction to DNA viruses
Laura Rayne
pg. 7
all. For that reason they need to synthesize all of the enzymes they need for DNA replication and
transcription so these are very large and complex viruses.
Slide 27: Variola, or smallpox, was erradicated in the 1970’s but there is still some concern that it may
be used as a bioterrorism agent. Vaccinia, or cowpox, was used as the first vaccine and was how variola
was eradicated in the first place. The concern is the emergence of a related virus in monkeys called
monkeypox, which can also infect humans and may cause serious disease. There are other pox viruses
for all kinds of other animals as well. In some cases these can also infect humans. The genomes of the
pox viruses are terminally redundant, partially double stranded DNA so they have this hairpin here.
Slide 28: This is a group of viruses. We are interested in the orthopoxviruses. Variola is the smallpox
virus and there are 2 forms of that. Vaccinia is the cowpox virus. Monkeypox, Camelpox. Some of these
viruses can infect humans but they don’t spread within the body. They only give localized infections,
except for possibly monkeypox. There is one other human virus, molluscum contagiosum, which doesn’t
give a very serious disease.
Slide 29: This is smallpox. One thing about smallpox is it spreads through the whole body. Variola
major is the most virulent type and causes hemorrhaging, internal bleeding and has 20-30% mortality
rates.
Slide 30: This is called orf. It is a poxvirus of sheep or goats. It can infect humans but gives rise to
localized lesions. They don’t spread through the body.
New slide: The replication cycle of vaccinia virus is complicated and quite interesting. The poxvirus is
this sort of brick-shaped virus. It has several layers of envelopes. It enters, gets uncoated, gives rise to
all these different transcripts and eventually sets up a replication center and then a viral factory, which
is where the virus is produced. When it assembles it first forms these things called crescents. Then the
replicated genome associates with this crescent, forming immature viruses. Then the ER-derived
membranes wrap around this and it matures into this brick-shaped thing that has 3-4 layers of
membranes.
Slide 31: This is what it looks like on electron micrographs. This is a replication complex here, which is
in the early phase of the replication when it is producing RNA transcripts but hasn’t started making virus
yet. Eventually this turns into a viral factory, which is this slightly lighter region. This is the nucleus
here. You can see some crescent shaped membranes. The dark dots here are the immature viruses,
which eventually give rise to the mature brick-shaped variants.
Slide 32: Skipped
Slide 33: I’m just going to sum this up. I’ve gone through a few viral groups and shown you a few
examples. I want you to think about what challenges the viruses face and what strategies they employ
to solve these challenges. For example, how does it get into the cell? How does it get into the nucleus?
How does it replicate its DNA? Does it use host polymerases? Does it have a circular genome? A linear
genome? What is it that the virus needs to replicate itself? What cellular functions can and does it use?
Microbiology: Introduction to DNA viruses
Laura Rayne
pg. 8
In some cases they bring in their own DNA polymerases, in some cases they don’t. Why is that? Where
does it find these functions? In most cases in the nucleus, except for the pox viruses, which need to
bring in their own functions for replication. Finally, for those that replicate in the nucleus, they can only
replicate in the S phase. It either has to only infect actively replicating cells, or it needs to provide
functions that will activate the cells, or it may go into a latent stage like herpes, or it can co-infect with a
virus that provides the converting function.