Wednesday Nite at the Lab.
I work here at the UW Madison Biotechnology Center.
I also work for UW Extension
behalf of those folks and our other partners, Wisconsin Public
Television, the Wisconsin Alumni
Alliance, thanks for coming to Association, and the Science
We do every Wednesday night, 50 Wednestimes a year.he Lab.
It's not always this gorgeous out, however, so I really
appreciate you coming tonight.
to introduce to you Holly Basta. Tonight I'm delighted to be able
PhD candidate in the cell and molecular biology program here
at UW Madison.
Ann Palmenberg's lab, and in She's a research assistant in
picornaviruses, pico standing for small and rna standing for
RNA.
that you'll get to hear more These are the small RNA viruses
Holly was born in Great Falls, about during Holly's talk.
If you've never been to Great Falls, I highly recommend it.
It's got a great Lewis and Clark
still pretty great. She completed her undergraduate
degree at Montana State University in Bozeman in 2008.
She just told me that she graduated, she got married, and
got in a car and moved here to
So it's all been on continuous, enjoyable series of research.
Tonight, she's going to talk
Vaccines: Insights From the abModel Structure in thed
Rhinovirus C. Please join me in welcoming
Holly to Wednesday Nite at the
[APPLAUSE]
Can everyone hear me okay?
Thank you all for coming to see my talk today.
I'm going to put in a plug for
things that have been going on vaccines because of a lot of
I'll get into my research. in the news lately, and then
today. I'm excited to talk to you
molecular biology is that DNA is So the central dogma of
protein. made into RNA that's made into
from this talk, you will know the central dogma of molecular
biology.
process called transcription, and then RNA leave the nucleus
of the cell and is then made
translation. into protein by a process called
talking about today are either So most of the things I'll be
So to get you, to introduce you to the jargon I'm going to be
using today, so when we're
the case of rhinovirus is thumans, the DNA is called thein
genome, the host genome.
and then that's translated into RNA is typicprotein.senger RNA,
When I'm talking about my rhinoviruses, their genetic
information is actually stored
DNA step, and that puts them a in RNA that completely skip the
So when I talk about their step ahead of the host.
and then when I talk about the genomes, I'm talking about RNA,
made by their RNA. capsid, that is a protein that's
Like I said, a virus can have either RNA or DNA, and that's
where all of their genetic
And then they have to have material is stored.
genetic material in, and that's scalled the capsid, and that'sr
just this shell of protein. What I'm showing you here is the
capsid cut in half. So it's basically a hollow globe
full of their genetic material. And throughout the talk I'm
going to introduce these
guys come away with better misconceptions in hopes that you
are. understanding of what viruses
run into is that viruses are a So the first misconception I've
absolutely not true. type of bacteria, and that is
different thing. Viruses are a completely
complex, but viruses are just this: a protein shell and RNA or
DNA.
people have is that viruses are nearly as big as human cells.
What I'm showing you here is a
So here's a human, a small scale of size.
Down here are things that you human, acan see. meter.
Thickness of human hair.
viruses are actually smaller Then visible light is here, and
And that's what make them so than that.
can't actually look at them underneath the light microscope.
And they're pretty much only
then atoms are down here. So I'm going to show you a coold
little schematic here.
Let's try this way.
We're actually going to skip
difficult. that one because it's being
So, this is a video. Alright.
has the flu. >> So let's say that this guys
them go. >> And I'm just going to let
sneeze containing, if we move in >> And here's a droplet from his
see each one of those little and take a really close look you
there are a lot of viruses purple things is a virus, and
of which go inevitably up this floating through the air, some
How did that guy feel you ripped unfortunate man's nose.
It was interesting because we did it while he was sleeping.
[LAUGHTER]
I am talking with medical illustrator David Bolinsky who
designed this video for a research company.
So here comes the virus, and
this guy's throat cells. it's going to land on one of
little yellow knobby things. So notice it's covered with
Those are the keys. >> This is a key..
This is a key.
Okay, if the keys on this virus happen to fit the locks, which
are the little purply stick-uppy
cell, if there is a match, the things on the surface of the
virus in. cell, wAnd what's this?mes the
This is the welcoming committee.
They all interlock with each
membrane down into the cell. other, and they pull this
deeper, and that welcoming >> And down it goes deeper and
virus capsule bursts and out structure disperses and the
to make more viruses, those comeslittle noodly things.r how
So this unsuspecting cell has been tricked into guiding these
virus recipes right into its own
So in they go. command center, the nucleus.
recognized by this big pink >molecule which his a miniy
factory.
It threads the nuclear >> What is it doing?
of the virus, through one hole, mand out another hole comes ae
brand new instruction set.
making copy after copy after >> So it's a copying machine
go out of the nucleus. copy of virus recipes which then
peanutty things, they cook up proteins that go back into the
nucleus where they are
and then out they go. They get covered up and head to
the surface where they get new
Here they come. This is an eruption of virus
after virus after virus after only virus entered this cell,
but how many came out?
Millions. So if one virus can produce a
million babies and do it again
this guy doesn't just drop dead? and again and again, how come
Well, because you have about a hundred trillion
cells.
a drop in the bucket when you >have a hundred trillion cells.
Remember, you do have your own
a virus usually... immune system which when it sees
So while the virus [LAUGHTER]
luck your immune system will does multiply fast, with any
So yes viruses, all viruses, work just a little faster.
That's what they do. want to spread.
them in check. But most of the time we do keep
Alright, so here's the thing Most of the time.
this. ISo this is actually a reallye
cool website. It's called
learn.genetics.Utah.edu, and
our movie is still making this has a lnoises.crolling bar,
So there's a little scrolling bar.
Now I restarted.
Alright, so there's a little I'm sorry.
it gets smaller and smaller. scrolling bar, and as you scroll
grain of rice. So here's a coffee bean and a
Grain of salt. Sesame seed.
getting to the microscopic And as you go in, now we're
Amoebas, paramecium, there's a things.
A photoreceptor in your eye. human egg and sperm.
Baker's yeast. Red blood cell.
E.coli. Mitochondria.
Now we're starting to see the viruses.
There's measles.
There's influenza. There's HIV.
rhinovirus. So it's really, really small,
and it's even small for a virus.
they showed you in the movie was And there's a ribosome, which
you get smaller and smaller there's an antibody all the ways
down to a single amino acid and then a carbon atom.
So that should give you some
are and how amazing they are to indcause so many problems.uses
Alright, so we watched that.
treat viruses. So there are two ways that we
vaccines. Both treatments are good in
their own ways. Drugs you're only given once
you're sick, and they're
that you have. specific to the specific virus
which is for the influenza The picture here is for Tamiflu,
They usually inhibit viral virus.
typically small molecules. development, and they're
carbons, usually, and they help only you versus vaccines which
help you and help others in that
people. So vaccines are given before
you're sick, as you know. They're specific to a certain
virus, and how they work is they
They say this is what this virus tip off the immune system.
When you see it, kill it. And so vaccines are typically
made from pieces or whole viruses.
So there's many different types of vaccines, and they range in
their safeness and effectivity. So not all vaccines are just
dead versions of viruses. So these attenuated and
inactivated viruses are, yes, dead versions of viruses.
Attenuation, they take out a
to be a full replicating virus. pAnd the vaccines that you mayds
have had that are attenuated
pox, or the nasal flu spray. viruses are the MMR, chicken
what they do is they shoot them with UV or they put them in ay
really highly acidic condition, and then the virus is dead or
inactivated.
made this way, and the flu shot One ofis made this way.es was
Fractionation, they essentially just chop up a virus, and then
you see little bits of the virus
infectious virus. buThe newer methods in makingly
vaccine are actually much safer because they take a piece of the
virus and then they put it into a bacteria's DNA, and the
bacteria actually makes the viral proteins, and then you
purify them out, and in that way
in contact with the vaccine that there's never a full virus ever
So one of the vaccines you may you get.
this are hepatitis B vaccine and have heard of that are made like
So the problems with these kinds the HPV vaccine.
know what the best part of the of vaccines is that you never
recognize is. vSo HPV requires three boosters
because it's not a really potent
So here's the vaccine schedule vaccine.
It's been a while since a lot of that the CDC recommends.
these are the ones that you get up until you're six years old.
So a major misconception is that
therefore untested, and what I'm most vaccines are new, and
these vaccines were around when showing yoI was a kid.at most of
A lot of them were around when
So influenza started in 1945. The Varicella is from 1974.
So a lot of these vaccines have been around for a long, long
time, and there's really strong data about how safe they are.
So I don't know if any of you heard of any of the news about
vaccines. Can anyone call out something
they've heard about a vaccine?
Autism. Autism is a big.
So I'm going to show you the
autism, and then you can make an actual data about vaccines and
and autism. So some of the other things that
people have concerns about are
and that actually was true but that vaccines contain mercury,
contain mercury. none of the current vaccines
amounts of mercury, and there were never any negative effects
noticed from them.
mercury in them. But currently no vaccines have
that doctors get paid to Other ones that I have heard are
That's false. Unless you know some dirty
doctors that I don't know.
biotech plots to get people I don'tvaccinated.e's any
So let's talk about vaccines and
So how they determine if autism.
is, essentially, you divide the sopeople who got, in this casee
autism, who had the vaccine by
didn't have the vaccine. the people who got autism that
relative risk. And this is how you calculate
there's no difference between Now relthe two groups.ne means
So the same amount of people who
the people who didn't get the got the vavaccines. autism as
So when they tested the MMR vaccine, which is the one that
was said to be associated with
people who didn't have the autism, actually slightly more
within 24 months it was pretty vaccine got autism, and then
And to put this into context, the relative risk of lung cancer
in smokers is over 20.
or no, you have even chances of So this getting autism.h vaccine
What about five years or ten
So the association, he asked years later?
later. what about five or ten years
within weeks or months after the The association was said to be
you get the vaccine at age two shot happened because many times
when autism symptoms cropped up. or three, I believe, and that's
originally go after this vaccine And that's what made them
So it just happened to be a coincidence that kids would
start developing autism around
vaccine. the same time they would get the
kids have shown to be autistic So, by later ages, typically
So then other myths are there's no risk to not getting
vaccinated and that vaccines can
So is it okay to not get wait.
So here are the MMR and DTaP vaccinated?
getting them. vSo one in a million people canf
get encephalitis or have a
these are the three diseases severe allergic reaction versus
Measles, one in 300 people die. that Mumps, one in 300 getinst.
encephalitis. And rubella, one in four can get
congenital rubella syndrome.
similar for the DTaP. There's been no proven deaths
from it, but one in 20 people die from diphtheria.
And it's easy to forget that
but actually now because a lot these diseases were a big deal,
vaccinate, a lot of these illnesses are making a comeback.
So these are pretty recent news articles.
This one says there's been 640
March 31st compared to 94 during cases of whooping cough as of
And this one is an outbreak of a the same time period last year.
single school district there meningococcal disease, and in a
actually died. were five cases and two students
that the measles cases were at a And last year the CDC reported
being available. 15-year high despite vaccines
cough epidemic at the moment >> Can you speak to the whooping
So, yes, there is a whooping going on?
moment. There is a combination of issues
between children get vaccinated very young for whooping cough
and sometimes the vaccines wear off, but that's also being
perpetuated by not being
So I'm just going to talk about herd immunity right now.
You lead me right into it.
people that need to be immunized So there's certain number of
from having a disease, and it's different for different viruses.
So, essentially, if 83% of
immunized to small pox, small pox won't be able to spread.
And so the problem right now is that these numbers are dipping
below, number of immunized
numbers, and we're losing our people aherd immunity.ow these
And then another misconception, since we're talking about polio,
is a lot of people think polio
need to be vaccinated anymore, is eradicated and there's no
In a lot of places it's impossible to get the polio
vaccine.
So the vaccine always has to It requires a cold chain.
the world that you just can't sbring something cool to them.in
So there's actually polio still cropping up in many places.
So we still need a vaccine. >> What about small pox?
Small is eradicated Small pox hasn't cropped up again.
So the only thing they're worried about small pox is if
it's in somebody's freezer
were lucky in that it doesn't shave an animal host so there's
nowhere for it to come from except for other people and
nobody has it anymore.
eradicated. S>> But if it should come back,y
they have not been giving small pox vaccines for quite a while.
That's correct. We haven't been vaccinated for
small pox in many years.
everything? SI just told you that vaccinesor
were awesome and everybody
So there's big problem with safety, and particularly with
the rhinovirus, which I'll talk about later, it definitely can't
have side effects that are worse
So the common cold isn't so bad in the spectrum of viruses, so
it has to have very, very minimal side effects to be
considered. So to get a safe vaccine, you
have to make sure that it actually does what it's supposed
to and that it alerts your
It has to be biologically stable, like I was talking about
with the polio vaccine. It has to be able to be
delivered to people in Third
practical delivery system. World countries and have a
system would be 10 shots over So an three months.elivery
Nobody is going to go in for 10 booster shots.
And the public must see more
actually become the biggest issue in recent years because
when I said vaccines, you guys
news story linking vaccines and sautism but I don't know thate
anybody saw that data that
And there's also been a lot of refuted it.
because people have moral issues issues with the HPV vaccine
That's all I'm going to say. with it.
mood, I have heard that people And then to kind of lighten the
and actually there isn't just so believe there's a zombie virus,
Not yet. you know.
So major challenges in making vaccines.]
Viruses have a very high
So, to put into context, you and mutation rate.
our lifetime and they may be I may have a couple kids over
from us, but a virus, like you slightly genetically different
billions of offspring, and each of them can have different
mutations, and they replicate within hours, and in that way
they're evolutionary time is
DNA viruses can integrate and associated with the host genome,
and so rhinoviruses don't do
actually shove themselves into this but some DNA viruses can
makes the virus right along with your DNA so your host machinery
Which ones are those? >> Retroviruses do that, like
HIV.
immune privileged regions of the So viruses can also infect
your nervous system and your host, and what I mean by that is
immune response whatsoever. Places like you airway and your
intestinal tract have great immune responses because things
from the outside come in and you
But many times viruses can find their way to your nervous system
which have not a very good
And then viruses have ways to get around the immune system by
directly shutting down parts of your immune system or sneaking
around it.
With rhinoviruses there's multiple serotypes, and the same
thing with influenza. So the reason you have to get an
influenza vaccine every year is it's a cocktail of many
different serotypes of
And then it's very expensive and influenza.
vaccines. Years and years and millions and
millions of dollars.
my research. So now we're going to talk about
of the common cold. RThere's over a hundred uniquee
serotypes, and they're divided
They're really small, simple into three species.
They're much smaller than HIV or viruses like I showed you.
So why don't we have a vaccine small pox.
We all get them. for rhinoviruses?
So rhinoviruses actually have a lot issues with developing the
vaccines that I told you about. They have a very high mutation
rate, and that's because they have the RNA genome that allows
them to be really fast and
They target the immune system, mutate quickly.
the cost and time involved in developing the vaccines becomes
an issue because there's not a
rhinoviruses. So the money goes into things
like HIV which a lot of people
So here's the schematic of the die of.
And remember your central dogma. rhinovirus genome.
It gets translated and these are This is the RNA.
And then the proteins get chopped up into smaller
proteins, and then they form
So here's a picture of a into a capsid.
of look at this like a 3-D map rhinovirus capsid, and we kind
specific landmarks that we know of the virabout.d there's
So here's, this triangle right
here. herSo all rhinoviruses have aon
canyon. So kind of a dip.
And then there's actually a hole
antiviral drugs are targeted to in go into this hole.the
And how they work is they essentially stick that hole
closed and then the virus is
So when it gets into the cell, trapped in its own capsid.
It can't release its genetic it's just stuck in the capsid.
more viruses. matSo then this is that sameake
triangle thing but these are
like. wSo the capsid is composed of a
VP1 which stands for viral
VP4 is inside the capsid. pSo these are all color coded.he
This blue is this blue over here.
So they have a five-fold, three-fold, two-fold, and
another three-fold axis.
repeated around into this So this triacapsid. essentially
Because they only have four proteins that make up the
capsid, it's repetitive.
capsid. So thSo what this is is aheir
phylogenetic tree, and we use these a lot to tell how closely
things are related to each
So these are all the known other.
So these ones right here are serotypes of rhinoviruses.
These right here are Cs, and rhinovirus As.
So these, the As, Bs, and Cs are these here are Bs.
other and make it a real all very different from each
vaccine or a single antiviral drug that will work on all of
them. And all of these strains are
circulating right now.
isolated at the hospital here. Most of these were actually
the one I'm interested in, and So the rhinovirus C species is
just recently discovered in the2007, but they found itwas
associated with almost half of
children. The reason they didn't find it
before is it can't be propagated
grow it now is they have to get iprimary cells from patients.
So if you have an operation on
snippet of those cells and then put rhinovirus on there and grow
it, but it's a really inefficient way to do it because
you have to count on people having surgeries and signing off
on their cells being used for things.
And then, oftentimes, those people are already infected with
a rhinovirus so it convolutes the results.
And then to grow these are any
interface system. So the reason for that is these
rhinoviruses grow in your airway
partly in air and partly in your and those cells like to be
So they're very difficult to mucus, essentially.
So what we normally use for cell grow.
here's what they look like. cAnd we grow them in little
dishes like this in serum.
virus has infected them is you And then how you tell if the
dead cells will appear as holes. cover them with this overlay and
been killed by the virus. So this is how we normally study
viruses, but because
cells, we weren't able to study rhinoviruses them. infect these
So the rhinovirus Cs are really interesting because they
actually have a higher
illnesses. propensity to cause more severe
from UW Hospital, and they So these are actually patients
with which rhinovirus different rpatients had, and they found
that rhinovirus C in red and
caused more severe illnesses rhinovirus A in black usually
are rhinovirus Bs. than the ones in green, which
that when they studied 128 And this separate group found
that rhinovirus C really children with asthma, they found
asthma much more than other exacerrhinoviruses.ldren's
So these viruses are very, very medically relevant, but we can't
grow them so it's very difficult
So why won't they grow? So in the video we saw, remember
they had the lock and the key?
about are actually host cell So the lock they were talking
your cell essentially says I receptors, and how these work is
environment, I'm going to put needout bait for it.the
And then when it picks up whatever it gets, it brings it
into the cell, into those balls that you saw in the movie, but
the viruses pretend like they're
says, oh, I have what I'm fishing for, and they pull it in
thinking they have what they
But we don't know what bait the rhinovirus Cs take, and we know.
it's not present in any of the
use. common cell culture lines we
We don't know what it is. So there's no way to get enough
virus to figure out this actual 3-D structure of what the Cs
look like.
computational methods, and we knew the 3-D structures of other
rhinoviruses and a lot of other proteins, I made the hypothesis
that I could use the known
methods to predict the structure structures and computational
And the method I used for that of rhinovirus Cs.
And so how that works is you is called 3-D protein threading.
whatever protein you want and you force it into differentf
confirmations.
confirmations of protein. And then with equations you find
out which is the lowest energy
fit into. struSo I did that with theuld
rhinovirus Cs, and then when I
structures of Bs and As, it ooverlapped pretty well.l
The only differences were in
the five-fold axis. this And so here's the fullh is
structures when I made them into
So here's real experimentally solved structures from A and B,
and here's the model I made.
looking. So they're pretty similar
closer, so right here, these are But when we went in and looked
tell because that has five the five-fold axis and you can
on that and cut it in half, we little points, when we zoom in
a very short five-fold axis. see that the rhinovirus Cs have
map projections, so how these And work is it's just like aoad
topographical map.
blue would be a valley. So here's the As, here's the Bs,
and here's the Cs.
axis where there's normally a Shill, there's actually no hill
in the Cs.
they're so drastically different And that's very interesting that
rhinoviruses. And this is very interesting
when you take into account that they don't use the same receptor
as the As and Bs. So this is the five-fold axis
and this is the five-fold axis. Both different types of
receptors that rhinoviruses use involve the five-fold axis.
And the rhinovirus Cs just don't
So they must be interacting with have it.
on their capsid. their receptor at somewhere else
rhinovirus Cs? So can we make a vaccine for the
vaccines is that you find the regions that are most likely to
confirm mutant protection after vaccination or natural
infection.
the rhinoviruses. AI don't think you can see itr
very well here. So here's these road mapped
projections.
your body sees the rhinovirus. And these are the regions that
at the five-fold axis. So right here, a lot of them are
body sees, but the major one is Tat the five-fold axis, and the
Cs are just missing those.
this is it makes loops of And hvariable regions.s with
So it says, oh, you're looking
Well this is going to be the at me here?
where I mutate the most. region where I vary the most and
have this spot to mutate. So there might be a possibilityt
that a vaccine would work better
evolutionary space. So major focus of my work was to
see if current antiviral drugs
So, like I told you before, drugs are typically designed for
this region here. It's called the hydrophobic
pocket. It has an open pore and a closed
toe.
pocket factors, so just pretty And normally in nature there are
in there, and they don't bind very tightly and allow the virus
to open up and release their
But drugs mimic these but bind genetic information.
capsid together. much more tightly and glue the
and here's it zoomed in, and So here's the five-fold axis,
sitting in the pocket. here is the antiviral drug
pretty much right up in here. So there are two antiviral drugs
that have come out of research
So there's pleconaril, and it was actually evaluated in two
large phase three clinical
work. It shortened the duration of
common cold virus and the window
It wasn't ultimately approved by that you spread the virus.
somehow interfered with birth control, and that's why it was
canned. But the next one that's coming
down the pike is called BTA.
strains, and an analog to this It inhibits 56 rhinovirus
clinical trials here at the drug righthospital.actually in
So there were two drugs I was interested in because they have
been the most effective so far
So we know which amino acids on rhinoviruses.
hydrophobic pocket, and this is a representation of those amino
acids.
besides the Cs fall into one So all of the rhinoviruses
One group of drugs works on one group of rhinoviruses and one
group of drugs work on another. So I wanted to know which group
the rhinovirus Cs fit into. So these are representation of
their sequences, and at lots of places they're the same.
So they all have the same amino acid here, but at some places it
differs a lot.
two drug groups and it's not in So this athese ones.s in these
So I then asked, well, which of these is actually important?
All of these amino acids are in the hydrophobic pocket, which
ones are most important for the drug binding?
So I analyzed all 27 of the structures that have been solved
for different rhinoviruses and
most, the drug was within four found that in all these cases
So what that means is these particular amino acids were
close to where the drugs was
with the drug. suggesting they were interacting
the ones that have been found And experimentally to bindare
pleconaril. So in this way I weighted which
amino acids were important for
So then I modeled in pleconaril into the different rhinoviruses,
and what I found was pleconaril fit just fine into the
rhinovirus Cs making me think that it would probably work on
them. So then through a collaboration
with a group at the hospital, they went and tested pleconaril
and this BTA 188, and they found
significantly decreased thrhinovirus C, which wasgs
interesting to us because my
these drugs would work. So then I went and looked a
little closer and found that the
is actually smaller in the Cs hothan it is in the As or Bs.et
So this is the As and this is the Bs, and they all have an
alanine which is a very small amino acid.
All of the rhinovirus Cs, however, have an isoleucine or a
similarly large amino acid. So when I simulated the alanine
being mutated to an isoleucine, you can see that it makes the
pore smaller. So what that made me think was
that yes maybe the drugs could
they probably couldn't get in fit onin the first place.in, but
And this particular amino acid
interesting because it's at the pore residue is very
enteroviruses. conserved within groups of
bigger group of viruses called So rhinovirus is a part of a
different groups either have the enteroviruses, and all the
bigger and it's conserved by the small alanine or something
So all bovine viruses don't have group.
enterovirus Cs have an alanine. So I thought that that was an
indication of how biologically
was. relevant this particular residue
rhinovirus C treatment? SMaybe a vaccine is possible.r
Probably not. They are only that one
immunogenic site so probably
mutate away from vaccines. they would still be able to
what type of drugs would fit But we now know a lot more about
might be that we need to develop into the rhinovirus Cs, and it
rhinovirus Cs. drugs specifically for the
the As or Bs will work for the None of the drugs that work on
So the good news is that we now have an idea what the rhinovirus
Cs look like, and we didn't have
primary cells. to grow them in anybody's
testing and we can also suggest receptors, and we've done that a
lot with the collaboration at the hospital.
Oh, we missed misconception.
[LAUGHTER] You can catch computer viruses.
some people think you can catch Icomputer viruses.d
So if you learn nothing else
your computer a virus and it from this talk, you can't give
So you should feel safe. can't give you a virus.
people that helped me with this So I'd like work.knowledge the
My lab is the Palmenberg lab, and we collaborated with -- for
the computational stuff and the Jim Gern lab at the hospital
helped us with testing the
And I'm funded by the NIH, and drugs.
[APPLAUSE]