Lecture 8: Viruses, RNA & Gene Expression
Slide 1 - Today we’re going to take a look at the interplay b/w RNA and the
organization of DNA.
- Start first of all with the novel aspect of the regulation of RNA done
by organisms that invade eucaryotes & procaryotes & that’s viruses
Slide 2 - Incidences of viral infection of animals such as the Avian flu –
starting with fowl-like poultry but then migrating to humans.
- Such flu viruses can have profound impact on populations of
individuals. Perhaps most profoundly in human population is the 1918
flu epidemic that killed 40 million people – more than both world wars
combined – wiped out almost a whole generation of people –
sometimes called the Spanish flu epidemic.
- Viruses don’t only infect animals but they infect everything from
procaryotes to eucaryotes.
Slide 3 - Here we see an example of viral infection of bacteria – we’ve got a
lawn of bacteria growing on surface of petri plate – all bacteria
growing right here in different titers or concentrations of viruses that
have been grown simultaneously with these bacteria. What we see as a
consequence are cleared areas and blacks where the viruses have
affected bacteria & lyse the cells & now infected bacteria, lyse the
cells & so on – see almost complete lyses at a high titer of the bacteria
at 10 .
- Such bacteria that infect microbes are known as bacteriophage.
Slide 4 - See it is infecting a bacterial cell – it’ll inject its genome into the cell
where it’ll be replicated, burst open & begin the replication cycle
again & we can see a bunch of T4 bacteriophage – very simple virus.
Missing Slide - There are others where we can see the exterior of the virus.
The capsid, a protein coat that surrounds - Poliovirus infects humans.
viruses - Tomato bushy stunt virus when it’s particularly difficult or causes
significant disease symptoms on tomato & related nightshades so even
Poliovirus tobacco, for example.
Tomato Bushy Stunt Virus - Tobacco mosaic virus – plant virus, creating symptoms on tobacco.
Tobacco Mosaic Virus - Looking at all of these viruses brings the attention that there is a
significant diversity in viruses that are out there.
Slide 5 - These viruses can be identified on the basis of their protein coat, the
capsid that surrounds the outside of the virus.
- Viruses can also be defined on the basis of the genome that they use,
that is RNA or DNA.
Slide 6 DNA
Slide 7 DNA
Slide 8 Host’s
- The way in which viruses replicate: viruses are so effective at what it
is they do b/c they’ve got an entirely paired down genome, very small
genome. What they do is they hijack or pirate the host’s gene
regulatory machinery (can see this in the figure).
- Figure: we’ve got a DNA virus with its protein coat, enters the cell,
releases its DNA, the DNA is transcribed by host’s transcriptional
apparatus, the DNA is replicated normally by enzymes provided by
the virus itself. The RNA is then translated using the host’s translational machinery & the DNA & protein get together to assemble
new viruses that are then released from the cell and the cycle begins
Slide 9 - Worked with a virus that has the capacity to infect tobacco.
- Virus infected tobacco (seen on the right) is all wrinkly & is less
efficient at what it does.
Slide 10 - Single-stranded RNA genome that encodes a # of different proteins.
- Encodes a replicase: RNA-dependent RNA polymerase (RdRp) –
must use RNA template to generate other RNA – this is what needs to
happen for an RNA virus – it needs to replicate its own RNA/genome
and that’s the enzyme that does it.
- In plants, the virus also needs to move from cell to cell through
plasmodesmata – small circular pore-like structures that are b/w plant
cells – movement proteins allow the virus to move from cell to cell
through plasmodesmata, at least in plants.
- Coat protein – makes up the capsid that protects the genome so when
it’s outside of the cell, that’s what’s going to be transmitted from one
plant to another – one host to another.
- RNA-binding protein – function not perfectly characterized today but
it’s thought that it may sequester the RNA to one part of cell in order
to ensure that it is translated.
Slide 11 RNA-dependent RNA polymerase (RdRp)
- 2 different plant cells and there plasmodesmata in b/w them from one
cell to the other. Let’s start with our RNA molecule.
Slide 12 RNA
Slide 13 5’ to 3’
- Binds to it & synthesize new RNA strand in the 5’ to 3’ direction
starting from the 3’ end of the template – so it functions just like a
- Note this important step: What’s happened is we’ve created a
double-stranded RNA molecule – this is very important (we will come
back to the importance of this in the future).
- Now it turns out that RdRp will now go back to synthesize a
complementary strand, but in the meantime, we’re going to talk about
what happens to that transcript? Where is it going to go?
Slide 15 Movement proteins
- Movement proteins binding to the plasmodesmata & we end up with
passage through the plasmodesmata so that they are now in another
- Just going to show the events occurring in the next cell but of course
they will be occurring everywhere that the movement proteins has
moved the RNA and of course in the original cell as well.
Slide 16 - Finally the virus will assemble, the co-protein will associate &
transmission, at least in plants, will occur by insect vector.
Slide 17 Self Assembly
- Identical coat proteins associate with each other without any outside
mechanism – it’s a form known as self assembly.
- Ask question that will be dealt with in the next lecture: you’ve got
such a cunning system that is got inside the host & has captured the
host’s mechanisms – how is it that hosts defending themselves against
such insidious invaders? **Lecture 8 finishes here **
**Lecture 9 starts here **
- In the past lecture he talked about viruses and how viruses captured or pirated components of the central
dogma as we see above, how they take advantage of the fact that the host effectively allows them to
transcribe and translate their own genomes so they can propagate from one generation to the next.
- What he promised to talked about was the interplay between this and the regulation of chromatin. Viruses
themselves don’t have chromatin so it must be something that happens to the chromatin of the host that’s
related to the virus.
Slide 18 Virus induced gene silencing
- He finished with the question: how do hosts defend themselves
against such insidious invaders? Ones that are taking advantage of
their own gene regulatory system and you can’t shut that down since
you have to regulate your own genes. How does an organism defend
itself against something that acts so stealthily? There are a number of
host defenses that can be invoked to protect the host against viral
pathogens, sometimes there are none. We know examples of this, for
example the acquired immune deficiency virus or the human
immunodeficiency virus HIV, there aren’t really effective host defense
- In other instances there is the adaptive immune response – the ability
to make antibodies that recognize the viral co-protein normally or the
replicase and thereby silence the virus through recognition of the virus
and shutting it down through the immune response.
- In addition there is something that is known as the innate immune
response, this isn’t derived from antibodies but are a series of
mechanisms inside the cell, and there is another form of immunity
(covered next lecture) which is not adaptive, that resides inside cells to
- We will touch on an innate immune response today; virus induced
gene silencing AKA RNA interference, it is one of the coolest recent
discoveries ever, it tells us how hosts protects themselves from viruses
highlighting a mechanism we end up using in just normal growth &
development to regulate gene expression.
Slide 19 - Back to hypothetical plant cells.
- Plant cells do not make antibodies, they don’t have the capacity to
make antibodies to protect themselves from viruses. Instead they
invoke virus induced gene silencing (VIGS) if they are able to.
Slide 20 - We have single stranded RNA in the plant cell, the RNA dependent
RNA polymerase comes along and synthesizes a new RNA strand.
- There is an important implication b/c in lecture what he said has been
done is that the RNA dependent RNA polymerase has created double
stranded RNA. This double stranded RNA is unusual for the cell, we
know there are instances of double stranded RNA & indeed we know
now that there are many instances of double stranded RNA. Ex: that
tRNA has regions of double strandedness as the tRNA molecule loops
back on itself and bonds then by Watson-crick base pairing through
complementary bases to make that T-like structure.
- Normally eucaryotic cell shouldn’t see double stranded RNA unless
invaded by a virus. Cells possess a surveillance system that detects
these double stranded RNAs and chops it up into 21-23 nucleotide
length fragments. Slide 21 The enzyme that does it is called DICER (RNase) which cuts inside
the RNA to create fragments of 21-23 nucleotide length fragments.
- This makes it effectively an endonuclease because it's cutting within
the RNA to give rise to the fragments.
Slide 22 RNA induced silencing complex (RISC)
- Multi-protein system is known as the RNA induced silencing
complex. This complex binds the 21-23 nucleotide fragments,
evidence suggests that it probably binds only one strand and this forms
a complex which contained within, is a small RNA segment that
corresponds to the original double stranded molecule. It will be
complementary to one or the other of those double strands so that’s
Slide 23 - What happens is that the RISC goes back and silences those
molecules that have sequence homology to these fragments – so it
goes back and recognizes portions of RNA and silences it. It prevents
it from being replicated and translated. It does this through two
mechanisms: one is to block translation and also it functions to
degrade the RNA (to say target this RNA specifically for degradation
– don’t use it because it's double stranded, it's bad most likely a virus).
Slide 24 - This silencing occurs throughout the plant body, the silencing system
travels through plasmodesmata (the tiny pores between cells) and
allows the defense mechanism to travel through the plant body. This is
said to be systemic silencing b/c it runs throughout the whole system.
Slide 25 - Here is everything. Now onto something that’s based on this.
Hopefully you’re thinking hey this is pretty cool, this system of the
recognition of double stranded RNA and silencing any RNA that is
homologous to what’s been recognized as being double stranded.
Slide 26 - Imagine that there is a mRNA that is synthesized from the host
genome (this is shown in the right cell, no nucleus in both cells to save
space but it's there really). The nucleus makes an mRNA (in blue).
Slide 27 - If you have a virus that has a part of that plant cell sequence
integrated into the viral genome (little bit of the messenger RNA
sequence from the plant has been integrated through genetic
engineering into the viral genome), it will be replicated and there will
be double stranded RNA where a segment of that RNA will
correspond to the mRNA originally derived from the plant genome.
- This is a genetic engineering experiment but it illustrates a very
important and cool point.
Slide 28 - This will be diced by DICER and recognized by RISC. What
happens as a consequence is that RISC travels through the plant body
looking to silence genes that bears homology to those 21-23
nucleotide sized fragments. It recognizes that one of those fragments
corresponds to the mRNA that was derived