David Baltimore (Cal Tech) Part 1: Introduction to Viruses and HIV

Uploaded by ibioseminars on 01.04.2010

Hello, I'm David Baltimore, President Emeritus of the California Institute of Technology
and now Professor of Biology at Caltech.
And, I'm going to talk today about HIV.
But I'm going to talk about it in the context
of what we know about viruses in general and where HIV came from,
and then go, particularly in the last two talks,
into therapeutic approaches and vaccine approaches to HIV
that are novel and that have been forced on us by the fact
that HIV has been so difficult to deal with
by standard methodologies.
In the first of the talks, I'm going to talk about a concept of equilibrium and non-equilibrium viruses,
and in particular describe how HIV can be considered non-equilibrium viruses,
like so many other viruses that are dangerous to us.
But let's first introduce viruses.
Viruses represent a separate kingdom of the living world.
Viruses are not like bacteria or fungi or other organisms
that can exist and grow as independent agents in the environment.
Viruses are very, very small, and so all they really have
is the genetic material to drive their duplication.
But, to actually carry out their duplication, they have to go inside of a cell.
And so, penetrating living cells becomes very important.
And then a virus is able to divert the cell's macromolecular synthesis
toward ends which are dictated by the virus.
Viruses have one of two kinds of genetic information -- either RNA or DNA.
This differentiates them from everything else in the living kingdom
that all use DNA as their genetic material...
that use DNA to send information from one generation to the next.
Viruses can use RNA. RNA and DNA are very similar to each other
chemically, but they're very distinct in how they are functioning
in biological systems, and so it's interesting and maybe even a remnant
of an earlier world that RNA can be a genetic material.
Viruses can grow inside cells of all sorts.
Animal cells, plant cells, even bacterial cells, and bacteria phages
were the first viruses that were studied by molecular biology.
But, if we're going to understand viruses, we've got to understand them in the context
of the central dogma of molecular biology.
And that central dogma, as first elucidated by Frances Crick,
says that DNA can duplicate itself... can make an exact copy of itself.
And so it can go from cell to cell in an exact copy.
The DNA in any given cell can be transcribed into RNA,
and that the RNA constellation of a given cell
is very important in terms of the specificity of that cell.
A skin cell has a different constellation of RNA than a liver cell or any other.
And finally, that the RNA encodes protein.
Proteins are the workhorses of viruses as they are of cells.
In 1970, I had the good fortune to make an observation
that added a piece to the central dogma, and that is reverse transcription.
Reverse transcription is the ability to reverse the ordinary flow of information,
so that RNA gives rise to DNA.
And that's something that was thought to be particular to viruses.
As we will discuss, it has much wider implications than that.
Now, since proteins are the workhorse of any biologic system,
the messenger RNAs that encode proteins
are the most important endpoint of a molecular system
that allows for controlling cell behavior.
And so, we can classify viruses by how they make their messenger RNA.
And in particular, there are DNA viruses and RNA viruses.
The DNA viruses come in 2 kinds. One kind has double-stranded DNA --
the standard kind of DNA that we find in the nucleus of all of our own cells
and the cells of other organisms in our environment.
So, for that double-stranded DNA to make messenger RNA is pretty straightforward.
There's a copying mechanism called DNA-dependent RNA polymerase
that copies DNA into RNA, and viruses either hijack
the cell's DNA-dependent RNA polymerase, or they bring in their own.
Then there are some viruses that have only one strand of DNA.
And those viruses have to duplicate that strand to make a double strand.
Then, that can be transcribed into message.
So, they have a somewhat more complicated life cycle.
Then, we have 4 kinds of RNA viruses.
RNA viruses that have double-stranded RNA as their genome,
and they have a polymerase that can copy double-stranded RNA
into single stranded messenger RNA.
Then there are viruses that have only the (+) strand,
which we call the messenger RNA or sense strand the (+) strand.
They have the (+) strand of RNA in their genome.
Viruses like the common cold virus are like that.
They get copied into (-) strand, the (-) strand becomes the template
for more messenger RNA... for more (+) strand.
Then there are viruses that have (-) strands of RNA as their genome.
And they get copied into messenger RNA in a direct fashion,
but of course, they have to be duplicated, so then they have to copy back the (+) strand
into the (-) strand.
And finally, we have viruses of the retrovirus kind
that have RNA as their genetic material, but copy that into DNA.
First into one strand, then into two strands,
and finally that can be transcribed into messenger RNA.
So, we can take all of the viruses in the world and put them in these 6 categories,
and that's a convenient way of thinking about them, at least for molecular biologists.
Viruses turn up in the news all the time.
HIV, which we'll talk about a lot, of course has been in the news
ever since its discovery in the early 1980s.
But, on the other hand, SARS appeared once in our environment,
as a potentially lethal virus. SARS came out of the animal world to
cause a relatively small number of deaths before we got it under control.
Smallpox is a virus that used to be rampant in our environment.
We've actually gotten rid of it, and as far as we know, it only exists in 2 places right now
In freezers in Russia and the United States.
Influenza is a virus that's continually in the news, because it keeps changing itself
and comes back every year in a different form
and now we're particularly worried about whether the avian flu will become a human flu.
There are some very famous incidents in which viruses just appeared out of the wild.
Marburg virus appeared in Germany. It turns out it had come from Africa,
killing a significant fraction of the people it infected.
Then later, about 10 years later, Ebola virus appeared.
Ebola is extremely lethal in that form, although there are other benign forms of Ebola.
Hantaan virus appeared in 4 corners of the United States, in the desert,
as a virus coming out of rodents...
Also extremely lethal to the relatively small number of people it infected.
And Norwalk virus is a very different sort of agent.
I put it on here because it turns up periodically in the news
when there's a cruise ship in which people get infected with a diarrheal virus.
That's norovirus, or Norwalk agent.
Now, I said that you need to understand molecular biology to understand viruses,
because they really are just molecular agents.
But, viruses also were very important in the history of molecular biology,
because many of the key experiments were done using viruses.
Hershey and Chase showed that DNA is the hereditary material.
They were actually rediscovering something that had been found with bacteria,
but it was their experiments that convinced the scientific world
that DNA was the hereditary material.
Luria and Delbrook showed that mutations pre-exist before evolutionary selection.
Meselson and Stahl showed that DNA replicated by copying of each strand into a duplex.
Herskey and Benzer showed that genes had fine structure.
And Brenner and company showed that messenger RNA existed at all
in experiments with viruses done at Caltech.
Molecular biology of animal cells has also been helped by understanding the behavior of viruses.
Splicing of nuclear RNA to generate messenger RNA was discovered in viruses.
We discovered reverse transcription, as I have described.
Plant viruses have been very important.
RNA was first shown to be a genetic material in plant viruses,
and that led to the idea which is now very current and very exciting
that perhaps the whole world that we see today
was once an RNA-based world... that DNA came later.
And finally, protection against pathogens by interfering RNAs was first shown with plant viruses.
Now, how many viruses are there?
There are literally an uncountable number of them.
And that's because every plant, every bacterium, every animal
has its own set of viruses, and we're never going to find them all.
We now recognize something like 1500+ species of viruses,
and each species has multiple kinds.
So, the numbers are already in the thousands, and can clearly be more than that.
How do viruses grow? We said the basic thing is they grown inside of cells.
They can multiply very fast. Once a virus gets inside a cell,
it may increase itself by 1000-fold in a few hours.
In fact, bacterial viruses can increase themselves by 100-fold in 20 minutes.
And, because viruses have to grow inside of cells,
they have to be passed from one host to another host to another host
to another host, continually. From me to you to somebody else to somebody else.
And if that chain is broken, the virus has lost its ability to function
in the environment.
And so, viruses specialize themselves to take advantage of the sociology of a species.
We get together in groups.. particularly,
our children get together in very intimate play games.
And so, viruses have taken advantage of that to spread among children,
from children to their parents, and of course, among adults also,
when we sneeze or cough or shake hands.
That's something we should think about changing.
So, if a virus is only a human virus, then if we vaccinate everybody,
so nobody is sensitive to the virus, for just a little while...
a year maybe, then the virus should be dead. It should have no place to hide.
And, in fact, that's true.
That's why we eradicated smallpox.
We virtually eradicated polio, but there are some places where it is still spreading.
Now, how do viruses get out of cells?
They develop inside cells.
They get out in one of two ways.
Either the virus can grow inside the cell.
Imagine this is the cytoplasm of the cell, that's the nucleus of the cell.
The virus can grown in here, and so if the virus can burst the cell open,
then it can just release itself. And some viruses do that.
They grow inside... bacterial viruses, in particular, mostly do this
They grow inside cells, cause the cells to break, and the virus is released.
But there's a way that viruses have to get out of cell that doesn't require killing the cell.
And that's to bud off the surface of the cell.
So, inside there, you have to imagine is the core of the virus.
The virus is coming out of the cell, pushing its way out, and taking modified cell membrane
that has virus-specific proteins in it out into the environment,
ultimately releasing itself as an infectious agent.
Now, as I've said before, viruses really only make sense in the context of molecular biology.
So, when there was no molecular biology,
(that is, before World War II, effectively)
it was impossible to understand the nature of viruses.
Viruses were just something small.
In fact, the definition of a virus was that it would pass through a filter that held back bacteria.
So, it's very small, but its genetic information is very powerful,
and it can make itself a coat, which is quite impervious to environmental stress.
Now, some viruses have a little more than a protective coat to them.
They hold inside themselves key elements that enable the virus to grow. We'll come back to that.
But, we couldn't understand any of this unless we had molecular biology.
Now, I said viruses are very small. The question you might have is, how do you see a virus?
Well, you can use the electron microscope.
The electron microscope has resolution that allows you to get down to nanometers,
and nanometers are the size of viruses.
Small viruses are about 20 nanometers in diameter,
large viruses can be many times that.
But, biologists developed a trick to visualize individual viruses,
which is, in fact, how all virus work was done for many, many years.
And that is to grow a monolayer of cells,
either animal cells or bacterial cells, or whatever is appropriate.
To take your suspension of virus... let's say it has 1 million particles per milliliter,
dilute it down so there are only a few particles,
and then put those particles on top of this monolayer.
Those particles will find cells to grow in, the cells will start to release virus,
it'll infect cells around them, and ultimately, you get a plaque.
And so you can count the number of infectious particles
by the number of plaques.
And if you've diluted the virus, you can figure out how many virus
you had in the original suspension by simply multiplying back.
These are plaques of bacterial viruses growing on a bacterial lawn.
These are plaques of polio virus growing on a lawn of human cancer cells.
Now, I said I was going to talk about equilibrium and non-equilibrium viruses.
Let me make that distinction here.
Equilibrium viruses are viruses that have been with a species for a very long time.
They have generally developed a modus vivendi with that species.
They're generally not lethal or not very lethal,
but they spread extremely well from animal to animal, person to person, bacterium to bacterium.
A very good example is the common cold virus.
We all know that if we come in contact with a person who has a cold,
we are likely to get a cold.
Somehow, that virus is going to transfer,
either through a sneeze or through physical contact.
So, these are equilibrium viruses in the sense
that they have developed an equilibrium status with their host.
They're not asking too much of their host... they're not going to kill their host, generally.
On the other hand, the host provides a way of transmitting
the virus on into generations in the future.
Now what's a non-equilibrium virus?
A non-equilibrium virus is one that's sort of jumped from another species,
where it is an equilibrium virus, into a new host organism
to which it is not well adapted.
Often, these viruses can be very lethal because they haven't been selected to be temperate.
They may spread very poorly, but sometimes they spread well,
and they represent most of the difficult problems that we have.
Most of the viruses on that slide that I showed you
are non-equilibrium viruses, like Marburg, and Ebola, and HIV.
And, HIV has developed the ability to spread well,
whereas something like Ebola virus spreads very poorly,
and in fact, tends to make its infected people so sick, so fast
that they only can transmit to very close family members
or to healthcare providers.
So, let's look at some equilibrium and non-equilibrium viruses.
Equilibrium viruses: Polio virus... almost eradicated.
Smallpox... eradicated. Common cold... there are too many kinds to eradicate it,
but it's a standard equilibrium virus. Measles, mumps, herpes...
lots of them. All actually, very specifically human viruses.
Non-equilibrium viruses: flu, HIV, SARS, Ebola, Hantaan...
viruses we've talked about already.
But, flu comes from birds, HIV comes from chimpanzees,
SARS and Ebola probably both come from bats,
Hantaan is an equilibrium virus in rodents.
So, let me turn now to HIV.
You will remember that the HIV epidemic started... or became evident to us
in the late 1970s. It became evident when people came down with a disease
called AIDS -- Acute ImmunoDeficiency Syndrome.
And what happened was, we first saw people who had this very severe immunodeficiency
and were dying of organisms that ordinarily shouldn't harm them.
Organisms that are around me and around you all the time.
and that are not much of a problem to us.
So, their immune systems somehow had totally failed
and couldn't handle the very simplest of challenges.
But, we didn't know what caused it, and it took a couple of years to figure that out.
And, you might remember that during that period of time,
people thought about the damnedest ideas.
They thought sperm caused it, they thought all sorts of things caused it
because it's sexually transmitted.
But, it turned out to be a virus... a virus which is sexually transmitted.
And that virus was then named Human Immunodeficiency Virus, or HIV.
How did we find HIV?
Actually, only when reverse transcription was discovered
would HIV have made any sense.
Now, we discovered reverse transcription around 1970. I'll come back to that.
And, HIV came around 1980, so we were lucky. We knew about reverse transcription,
we knew how to find HIV by assaying for reverse transcriptase.
But, imagine the reverse.
Imagine HIV came in 1970, and we hadn't yet discovered reverse transcriptase.
Well, in that case, it would have been very hard to find HIV
because there just isn't much of it around.
It's hard to see in the electron microscope.
It's hard to do anything but measure the enzymatic activity
of reverse transcriptase.
So, we were lucky in the order in which they came.
Let me go back to that discovery of reverse transcriptase
and put it in the context of my particular life in science.
So, in 1960, I entered the field of molecular biology. I was just out of college.
I was going to graduate school. We knew about 2 kinds of polymerases,
enzymes that can catalyze the synthesis of nucleic acid -- RNA and DNA.
One was the DNA polymerase, which we believed to be involved in replication,
and the other was the DNA-dependent RNA polymerase,
which was involved in gene transcription.
Then, in 1962, as I began to work on Polio virus,
I realized that it might well have an RNA-dependent RNA polymerase
and assayed for that and found in Polio virus-infected cells,
that there was such an enzyme, and now we know that all RNA viruses
encode enzymes of that sort.
Then in 1969, we realized that there was a class of negative-strand viruses,
viruses that had the (-) strand... the class... well, I can't remember what number it is,
but the class of viruses that have negative strands.
And, so we looked for a polymerase for that, and, in fact,
there was one. But, that was found in the virus particle,
not in the infected cells so much, so that said maybe virus particles have interesting enzymes.
Now, let's go back in time... 1960s.
Howard Temin then, just having gotten his degree at Caltech,
and going to the University of Wisconsin, speculated that a certain kind
of tumor-causing virus... cancer virus...
which carried RNA as its genetic material, could transfer that material into DNA.
And he suggested that because cancer-causing viruses cause permanent changes in cells.
They change cells from normally growing cells to cancerous cells that grow without control.
And so, they're doing something permanent to cells
but RNA generally in cells has a transient role.
Messenger RNA is degraded, other kinds of RNAs turn over.
They very rarely have a stable, information-carrying role,
except as I've said, in certain kinds of viruses.
So, he said, well, maybe RNA turns into DNA.
For 10 years, he had worked on trying to prove that.
Then, in 1970, Howard Temin and I independently discovered that in the virus particles
of RNA tumor viruses, there was an RNA-dependent DNA polymerase,
which was then dubbed reverse transcriptase,
because it reverses the standard flow of information in biological systems.
And, because that discovery had lots of implications,
in 1975, we shared the Nobel Prize in Physiology or Medicine for that discovery
with Renato Dulbecco, who had been a mentor to both of us,
and who had also done very important work on another class of viruses.
So, here's reverse transcriptase. Over here is a ribbon model... you can't see much.
Over here is a schematic model, where you can see a lot.
So, what you can see is that the RNA template comes in the top of the enzyme here.
That in here is the polymerization machinery,
that makes a copy into DNA, which is the red molecule,
that then continues further down in the reverse transcriptase,
where there's a little component called the ribonuclease H,
that recognizes that this is a hybrid between RNA and DNA
and degrades the RNA portion of the hybrid.
So, the genome that came in here is actually degraded right away, once it's been copied,
and out the other end comes a pure DNA strand
that then can be duplicated again by reverse transcriptase.
So, that was the discovery.
What have been the implications since that discovery in 1970?
Well, first of all, we changed the name of RNA tumor viruses.
They became retroviruses, indicating that they reverse the flow of information.
Secondly, and much more importantly, it was recognized that cancer can be caused
by the permanent association of new genes with a cell.
That that model was correct and the implication was
that cancer was due to mutations of genes, and in fact that has been proven to be true
in many, many cases of cancer in humans and other animals.
A third implication was that since we're copying RNA into DNA,
maybe you could copy messenger RNAs into DNA.
And so, we very quickly worked out a way to do that,
and that was one of the starts of biotechnologies, because in a sense,
what you're doing is capturing in a genetic form -- in double-strand DNA --
the information in a messenger RNA, and so you've found a way
to make a protein in very large amounts
using artificial DNA constructs that were copied from messenger RNA.
And that's what started biotechnology.
Something I, at least, was not at all prepared for was the discovery
that many mobile DNA elements -- genetic elements --
things that hop around in the genome,
first discovered by Barbara McClintock in plants...
That many of these use reverse transcription.
So, they copy a piece of DNA into RNA, the RNA is then copied back into DNA,
the DNA hops to another place in the genome.
That happens in the history of all species,
and in humans, it's happened so frequently, that some 45% of our genome
arose in this manner.
So, 45% of our genome came by basically reverse transcription.
And finally, relevant to this discussion, is that HIV was found to be a retrovirus,
something that was discovered only 10 years after the discovery of the polymerase.
So now we can put together a life cycle for HIV.
HIV, as a free agent, has reverse transcriptase inside of it.
It has some other things inside of it that we'll come back to.
It has on the outside of it, little spikes, and those spikes
recognize the surface of certain kinds of cells.
We'll come back to which cells in a minute.
And, fuse with the surface of that cell, putting the inner components of the virus
into the cell. There, reverse transcriptase gets activated,
and it now make a double-strand DNA copy of the RNA genetic information.
That then, because there's an integrase protein in here also,
that can integrate into the host chromosome.
It goes into the nucleus and integrates in the host's chromosome.
And, that then encodes messenger RNA.
Because it's DNA, it can be read by DNA-dependent RNA polymerase,
makes messenger RNA, and makes new genomic RNA.
Messenger RNA encodes viral proteins. The cell gets viral proteins on its surface,
as well as inside it. You get budding out of the sort that I mentioned before,
and you make new virus particles.
That budding process is illustrated in this slide
Actually, this is not HIV. It's another virus. I think measles.
But, you can see very nicely how it buds off the surface, carrying with it a coat of protein
underneath, or actually, going through the membrane.
Carries the genetic material inside it, and ultimately,
it'll pinch off here and release a free virus particle.
Now, with HIV, the virus particle has a very strange property.
And that is that this, what we call nucleoid inside the particle
is not symmetric. And in almost all the viruses, this is spherically symmetric.
Here, it's this asymmetric structure, for reasons that are not clear,
but represent a signature of HIV.
So, this is a nice diagram of the HIV particle.
You can see the spikes on the outside -- this is what attaches to cells.
You can see the genome, which is actually two molecules of RNA.
You can see the reverse transcriptase in here.
You can see the integrase, which is going to help
the double-stranded DNA go into the chromosome.
And you can see another enzyme, a protease, that's important for maturing the virus.
Now, you can't talk about HIV, without talking about how awful
the HIV epidemic and the AIDS epidemic has been.
65 million people have been infected at this point.
The data is actually 2005 data.
25 million people dead, 40 million people living with AIDS.
5 million of those in India, and about another million in China
as far as we know, and I call those out, because those are the most populous countries in the world,
and they are spreading the virus.
Today, there are about 13,000 infections per day
mostly still in Africa -- 5 million a year.
3+ million people dying every year of the viral infection.
That's as much as the 2 other infections that have been a scourge
for many years of the less developed world -- tuberculosis and malaria.
The effect on African countries has been dramatic and awful,
In some countries, seeing a reduction in average lifespan of as much as 20 or more years.
So, why is this so lethal?
Fundamentally, it is a classic non-equilibrium virus.
Actually, HIV (or a very close relative of HIV, almost certainly its parental virus)
is endemic in Africa in great apes, found in chimpanzees,
and it got a foothold in the human population, some significant length of time ago.
Some people say as much as 70 years ago.
Probably, it was spread at very low levels, maybe very inefficiently,
and not recognized because it doesn't cause a characteristic disease.
It causes things that look like a lot of other things.
And, then it came to the United States, probably on an airplane
in the 1970s and started killing people in numbers
that were large enough that they were recognized by physicians
in the late 1970s.
So, why is HIV lethal?
Well, it grows in one of the key cell types of our immune system.
So, our immune system has lots of different cells in it,
but one of them is a helper cell that helps everything else take place.
If you don't have helper cells, the immune system is pretty puny.
And, HIV has targeted itself on helper cells.
So, infected people lose their helper T cells over years
because it takes that long for the virus to get through the cells.
And ultimately, they become immunodeficient, and they die from infections
that are caused by organisms that healthy people would ordinarily just get rid of.
And so, I've taken us from a very general consideration of viruses
to the very specific virus, HIV, a virus that grows in helper T cells
and causes lethality in humans -- a virus that we have to stop,
and yet we have found it very difficult to stop its transmission.
Why is that true?
That will be the subject of the discussion in our next two parts. Thank you.