Discovering the Unique Genetic Systems of RNA Viruses: From Picornaviruses to Retroviruses | Study notes Virology

VIRUSES, POLYMERASES AND CANCER

Nobel Lecture, December 12, 1975

DAVID BALTIMORE

Massachusetts Institute of Technology, Cambridge, Mass., U.S.A.

The study of biology is partly an exercise in natural esthetics. We derive

much of our pleasure as biologists from the continuing realization of how

economical, elegant and intelligent are the accidents of evolution that have

been maintained by selection. A virologist is among the luckiest of biologists

because he can see into his chosen pet down to the details of all of its mole-

cules. The virologist sees how an extreme parasite functions using just the most

fundamental aspects of biological behavior.

A virus is a form of life with very simple requirements (1). The basic

needs of a virus are a nucleic acid to be transmitted from generation to gen-

eration (the genome) and a messenger RNA to direct the synthesis of viral

proteins. The critical viral proteins that the messenger RNA must encode are

those that coat the genome and those that help replicate the genome. One of

the great surprises of modern virology has been the discovery of the variety of

genetic systems that viruses have evolved to satisfy their needs. Among the

animal viruses, at least 6 totally different solutions to the basic requirements

of a virus have been found (2).

If we look back to virology books of 15 years ago, we find no appreciation

yet for the variety of viral genetic systems used by RNA viruses (3). Since

then, the various systems have come into focus, the last to be recognized

being that of the retroviruses (“RNA tumor viruses”). As each new genetic

system was discovered, it was often the identification of an RNA or a DNA

polymerase that could be responsible for the synthesis of virus-specific

nuclei acids that gave the most convincing evidence for the existence of the

new system.

Now that the life-styles of different types of viruses have been delineated

we can ask what relation there is between a virus’ multiplication cycle and

the disease it causes. In general, this question has no simple answer because

disease symptoms do not correlate with the biochemical class of the virus. For

instance, both poliovirus and rhinovirus are picornaviruses but one causes

an intestinal infection with paralysis while the other causes the common

cold. One class of RNA viruses, however, does have a unique symptom asso-

ciated with it: the biochemically-defined group of viruses called the retrovi-

ruses are the only RNA viruses known to cause cancer. For a virologist interest-

ed in cancer, the problem is to first understand the molecular biology of

retroviruses and then to understand how they cause the disease.

In what follows, I will review my personal involvement in uncovering the

different genetic systems of RNA viruses, a story which leads to the recogni-

tion of the unique style of retroviruses. I will then consider what is known

215

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VIRUSES, POLYMERASES AND CANCER

Nobel Lecture, December 12, 1975

by

D A V I D B A L T I M O R E

Massachusetts Institute of Technology, Cambridge, Mass., U.S.A.

The study of biology is partly an exercise in natural esthetics. We derive

much of our pleasure as biologists from the continuing realization of how

economical, elegant and intelligent are the accidents of evolution that have

been maintained by selection. A virologist is among the luckiest of biologists

because he can see into his chosen pet down to the details of all of its mole-

cules. The virologist sees how an extreme parasite functions using just the most

fundamental aspects of biological behavior.

A virus is a form of life with very simple requirements (1). The basic

needs of a virus are a nucleic acid to be transmitted from generation to gen-

eration (the genome) and a messenger RNA to direct the synthesis of viral

proteins. The critical viral proteins that the messenger RNA must encode are

those that coat the genome and those that help replicate the genome. One of

the great surprises of modern virology has been the discovery of the variety of

genetic systems that viruses have evolved to satisfy their needs. Among the

animal viruses, at least 6 totally different solutions to the basic requirements

of a virus have been found (2).

If we look back to virology books of 15 years ago, we find no appreciation

yet for the variety of viral genetic systems used by RNA viruses (3). Since

then, the various systems have come into focus, the last to be recognized

being that of the retroviruses (“RNA tumor viruses”). As each new genetic

system was discovered, it was often the identification of an RNA or a DNA

polymerase that could be responsible for the synthesis of virus-specific

nuclei acids that gave the most convincing evidence for the existence of the

new system.

Now that the life-styles of different types of viruses have been delineated

we can ask what relation there is between a virus’ multiplication cycle and

the disease it causes. In general, this question has no simple answer because

disease symptoms do not correlate with the biochemical class of the virus. For

instance, both poliovirus and rhinovirus are picornaviruses but one causes

an intestinal infection with paralysis while the other causes the common

cold. One class of RNA viruses, however, does have a unique symptom asso-

ciated with it: the biochemically-defined group of viruses called the retrovi-

ruses are the only RNA viruses known to cause cancer. For a virologist interest-

ed in cancer, the problem is to first understand the molecular biology of

retroviruses and then to understand how they cause the disease.

In what follows, I will review my personal involvement in uncovering the

different genetic systems of RNA viruses, a story which leads to the recogni-

tion of the unique style of retroviruses. I will then consider what is known

216 Physiology or Medicine 1975

about the relationship between the biochemistry of retroviruses and their abil-

ity to be oncogenic.

As I tell my story I will mention a few of the many co-workers, teachers and

students who have influenced my thinking or contributed their labors and

ideas to the products of my laboratory. To mention all of the people to whom

I am indebted would make too long a list; I can only say that the honors I re-

ceive are in large measure testaments to their accomplishments.

P I C O R N A V I R U S E S

My work on the genetic systems of RNA viruses dates back to my graduate

school days. As part of my introduction to animal virology during a Cold

Spring Harbor course, I heard Richard Franklin describe his then-recent ex-

periments using autoradiography to show that Mengovirus, a picornavirus

and a close relative of poliovirus, could shut off the nuclear synthesis of cellu-

lar RNA early after infection and could later induce new RNA synthesis in

the cytoplasm which appeared to represent synthesis of viral RNA (4). I decid-

ed to go to the Rockefeller University as a graduate student with Richard

Franklin in order to work on the system he had developed.

Before I began to study how picornavirus RNA was made, it was already

known from the work of Simon that picornavirus RNA synthesis was inde-

pendent of DNA synthesis (5). Furthermore, studies with actinomycin D had

shown that neither synthesis nor expression of cellular DNA was involved in

viral RNA synthesis (6). These results suggested that Mengovirus might

make a cytoplasmic RNA-dependent RNA synthesis system. The concept that

viruses induce synthesis of their own enzymes had strong precedents in bacte-

riophage systems - Seymour Cohen’s work had shown decisively that new vi-

rus-specified enzymes were found in infected bacteria (7).

We approached the problem of the virus’ effect on intracellular RNA syn-

thesis as a question in enzymology. We first showed that the nuclei from

Mengovirus-infected cells were greatly reduced in their ability to carry out

cell-free DNA-dependent RNA synthesis compared to nuclei of uninfected

cells (8). Later, we showed that cytoplasmic extracts of Mengovirus - or

poliovirus-infected cells contained an RNA synthesis activity not evident in

uninfected cells and not inhibited by actinomycin D (9). When we learned

that the system made RNA of the size and structure of virion RNA (10), it

became clear that it represented the postulated viral RNA-dependent RNA

synthesis system.

While there has been extensive further analysis of crude cytoplasmic sys-

tems ( 11, 12) and impressive enrichment of the RNA synthesis system has

been achieved (13),. no pure enzyme able to make picornavirus RNA has ever

been isolated so the detailed mechanism of viral RNA synthesis still remains

obscure. Most of our knowledge of how picornavirus RNA is made has come

from studies on the virus-specific RNA molecules in infected cells and their

kinetics of labeling by radioactive precursors. Such research has been carried

out in many laboratories ( 11, 12); my work in this area was done in associa-

tion with James Darnell and Marc Girard. Together we found and studied

218 Physiology or Medicine 1975

RNA synthesizes a “minus” strand which in turn synthesizes a series of plus

strands. This diagrammatic simplicity of poliovirus replication hides a fair

amount of as yet undeciphered complexity as shown by the work of Eckard

Wimmer and his colleagues as well as by work in my laboratory. For instance,

the 3’-ends of the virion RNA and messenger RNA are both poly(A), the 5’-

end of the minus strand is poly (U), so we assume that they are templates for

each other (21). But these homopolymer stretches have very variable lengths

even in the progeny of a cloned virus; what then determines their length? The

poly (A) serves some obscure but necessary function in the life-cycle of the vi-

rus (22); what is this function? There is no triphosphate 5’-terminus, either

free or capped, on the virion RNA or messenger RNA (23, 24); how then is

the synthesis of these molecules initiated? The 5’-end of the virion RNA and

messenger RNA are different (24); what does this mean?

V E S I C U L A R S T O M A T I T I S V I R U S

Most of the work in my laboratory until 1969 centered on poliovirus. We had

assumed that all RNA viruses would be similar in their basic molecular biol-

ogy but during the 1960’s results emerging from various laboratories implied

that poliovirus, rather than being a model for all RNA viruses, used one out

of a collection of different viral genetic systems. Probably the first dramatic

demonstration of the variety in RNA viruses came from next door to Richard

Franklin’s laboratory at the Rockefeller Institute where Peter Gomatos and

Igor Tamm found that reovirus has double-stranded RNA as its genome (25).

The peculiarity of reovirus was underscored by the demonstration later that

an RNA polymerase in the virion of reovirus is able to a symmetrically tran-

scribe the double-stranded RNA (26). Th is was the first virion-bound RNA-

dependent RNA polymerase ever found and followed after the finding of the

first nucleic acid polymerase in a virion - the DNA-dependent RNA polymer-

ase found by Rates and McAuslan and Munyon et al in virions of vaccinia

virus (27).

Another observation that suggested there were profound differences among

the RNA viruses was the finding that in cells infected by the paramyxovi-

ruses, Newcastle disease virus or Sendai virus, much of the virus-specific RNA

was complementary to the virion RNA (28). This result was in sharp con-

trast to what was found in poliovirus-infected cells where most of the virus-

specific RNA was of the same polarity as virion RNA (11).

We branched away from concentration only on poliovirus to include the

study of vesicular stomatitis virus (VSV) because of the lucky circumstances

that brought Alice Huang to my laboratory. She joined me first at the Salk

Institute and then we both came to M.I.T. in 1968. Alice had studied VSV as

her graduate work with Robert R. Wagner at Johns Hopkins. We decided

that the techniques developed for studying poliovirus should be applied to

VSV, hoping that the peculiar ability of VSV to spawn and then be inhibit-

ed by short, defective particles could be understood at the molecular level. A

graduate student, Martha Stampfer, joined in this work and together we

Viruses, Polymerase and Cancer 219

found that we had bitten off an enormous problem because VSV induced

synthesis of so many species of RNA. In poliovirus-infected cells, only three

species of RNA are seen but we found at least 9 RNA’s in VSV-infected cells

and one of these RNA’s was clearly heterogeneous (29) - later work showed it

had 4 components (30, 31). In our description of this work we said that 9

RNA species “seems exorbitant” (29) but we soon realized that each RNA

had its place in the cycle of growth and growth inhibition of VSV.

As we were beginning to unravel the multiple VSV RNA’s, Schaeffer et al

(32) published a paper showing that the major VSV-induced RNA’s in in-

fected cells, like those induced by Sendai and Newcastle disease viruses, were

complementary in base sequence to the virion RNA. We confirmed and ex-

tended that observation, showing that the virus-specific RNA recovered

from the polyribosomes of infected cells (the viral messenger RNA) was all

complementary to virion RNA (33). This finding presented a pregnant para-

dox: if all viruses were like poliovirus and induced a new polymerase in the

infected cell how could a virus that carried as its genome the strand of RNA

complementary to messenger RNA ever start an infection? There seemed two

possible solutions: the RNA came into the cells and was copied by a cellular

enzyme to make the messenger RNA to initiate the infection cycle or the

RNA came into the cell carrying an RNA polymerase with it.

Because no convincing evidence for RNA to RNA transcription existed

(or exists) for any uninfected cell, the possibility of a polymerase with the

incoming RNA seemed attractive. This possibility implied that there might

be polymerase activity demonstrable in disrupted virions of VSV. Almost

as soon as the power of this reasoning was clear to us, we had shown the

existence of the virion RNA polymerase (34). The demonstration of this en-

zyme was the piece of evidence that led to the realization that there is a huge

class of viruses, now called negative strand viruses (35), that all carry the

strand of RNA complementary to the messenger RNA as their genome and

that carry an RNA polymerase able to copy the genome RNA to form multiple

messenger RNA’s,

R E T R O V I R U S E S

The discovery of a virion polymerase in VSV led us to search for such en-

zymes in other viruses. Because Newcastle disease virus made a lot of comple-

mentary RNA after infection it seemed a logical candidate and after an ini-

tial failure (34), we found activity in virions of that virus (36). But a

more exciting possibility occurred to me; maybe by looking for a virion poly-

merase, light could be shed on the puzzle of how RNA tumor viruses multiply.

In his Nobel lecture, Howard Temin has outlined how he was led to postu-

late a DNA intermediate in the growth of RNA tumor viruses (37). Al-

though his logic was persuasive, and seems in retrospect to have been flawless,

in 1970 there were few advocates and many skeptics. Luckily, I had no ex-

perience in the field and so no axe to grind - I also had enormous respect for

Howard dating back to my high school days when he had been the guru of the

Summer School I attended at the Jackson Laboratory in Maine. So I decided

Viruses, Polymerase and Cancer^221

Expression of the Integrated

Figure 2. The life cycle of an RNA tumor virus. Based on present knowledge (42), the life cycle of an RNA tumor virus can be separated into two parts. In the first part the virion attaches to the cell and somehow allows its RNA along with reverse transcriptase to get into the cell’s cytoplasm. There the reverse transcriptase causes the synthesis of a DNA copy of the viral RNA. A fraction of the DNA can be recovered as closed, circular DNA (43) and it is presumably that form. which integrates into the cellular DNA. Once the proviral DNA is integrated into cellu- lar DNA it can then be expressed by the normal process of transcription. The two types of product which have been characterized are new virion RNA and messenger RNA. Much of the messenger RNA which specifies the sequence of viral protein is of the same length as the virion RNA but there may also be shorter messenger RNA’s (48). The vi- rus-specific proteins have 2 known functions: one is the transformation of cells which occurs when, for instance, a sarcoma virus infects a fibroblast, the second is to provide the protein for new virion production. The transforming protein is shown here as acting at the cell surface but that is only a hypothesis.

viral proteins made in the infected cell may be a product that changes the

growth properties of the cell (50); in such a case the retrovirus becomes a tu-

mor virus.

The second period of the infection cycle can be dissociated from the first

in a number of experimental systems. For instance, mammalian cells infected

by avian viruses can gain viral DNA but not express it (46). Also, cells can

have viral genomes that they inherited from their ancestors and such ge-

nomes are generally not being transcribed. Nonexpressed genomes can be acti-

vated: bromodeoxyuridine and iododeoxyuridine, for instance, stimulate the

expression of inherited, silent viral genomes (51). The exact mechanism of

activation of the genome for transcription, initiation of the transcript and

termination of transcription are obscure, as are any processing events of the

initial transcript which may occur.

222 Physiology or Medicine 1975

It is evident that the key piece of machinery provided by the virus for this

unique life cycle is the reverse transcriptase. Purified reverse transcriptase has

the properties of most DNA polymerases: it is a primer-dependent enzyme

that makes DNA in a 5’ 3’ direction using deoxyribonucleoside triphos-

phates as substrates and taking the direction of a template for determining the

base sequence of the product. The enzyme differs from normal cellular DNA

polymerases by having a unique polypeptide structure, having an associated

ribonuclease H activity and being able to make copies of RNA templates as

readily as DNA templates (41). Genetics has shown us that the avian leuko-

sis viral enzyme, at least, is encoded by viral RNA and needed only in the

first period of the infection cycle (52).

The primer-dependence of the reverse transcriptase means that the enzyme

can only elongate nucleic acid molecules, it cannot initiate DNA synthesis de

novo. How then does the enzyme initiate the copying of viral RNA? The an-

swer is that the genome RNA has attached to it a primer RNA molecule

which is, in the case of avian leukosis viruses, cellular tryptophan transfer

RNA (53). The avian leukosis virus reverse transcriptase has a high-affinity

binding site for that transfer RNA which the enzyme presumably uses for

precise initiation of reverse transcription (54).

RETROVIRUSES AND CANCER

The last 15 years of research in animal virology has allowed us to see the di-

versity of genetic systems used by the various types of RNA viruses and has

most recently shown us how distinct the retroviruses are from the others.

Rather than using an entirely RNA-dependent replication and transcription

machinery, the retroviruses have included the DNA provirus in their life-

cycle. Having a DNA intermediate does not make their mode of growth espe-

cially complicated - the DNA formally takes the place of the “minus” strand

in the picomavirus genetic system - but the DNA is probably the clue to why

retroviruses are the only ones able to cause cancer. The DNA provides the nec-

essary stability to the virus-cell interaction so that a viral gene product can per-

manently change the growth properties of an infected cell. Equally signifi-

cant, the DNA stage is probably important to the ease with which retrovi-

ruses carry out genetic recombination (55) ; it is quite possible that the recom-

bination system can bring together host cell genetic information with viral

information and that in this way non-oncogenic retroviruses become oncogen-

ic (56).

So the inclusion of a proviral stage in the retrovirus life-cycle may provide

critical capabilities towards the development of an oncogenic potential. But

the actual transformation of cells by retroviruses is a highly selective process;

each type of oncogenic virus transforms a very limited range of cell types (57).

If we assume that all transforming genes of viruses are like those of Rous

sarcoma virus, genes that are not necessary to the growth of the virus (50, 58),

then we can postulate that each type of transforming virus makes a specific

type of transforming protein. Such a protein, by this model, would not be

224 Physiology or Medicine 1975

and cellular activities and does not require the ad hoc postulation of beneficial

properties of viral products. It treats retroviruses like any other virus, as an

entity with its own life-style and its own accomodation with evolution.

In summary, I have tried here to develop the view of retroviruses as one of

a number of solutions to the problem of creating a virus. Each virus directs

synthesis of two critical classes of proteins: proteins for replication and

proteins for constructing the virus particle. By encoding the reverse tran-

scriptase, retroviruses have evolved the ability to integrate themselves into the

cell chromosome as a provirus. This is a very sheltered environment in which

to live, only mutation interferes with the continual transmission of the virus

to the progeny of an animal that is infected in its germ cells. In this context,

the ability of some retroviruses to cause cancer is a gratuitous one. But it is

today the most challenging and important attribute of these retroviruses and

the one that will dominate future research efforts in this area.

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Discovering the Unique Genetic Systems of RNA Viruses: From Picornaviruses to Retroviruses, Study notes of Virology

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VIRUSES, POLYMERASES AND CANCER

Nobel Lecture, December 12, 1975

by

D A V I D B A L T I M O R E

Massachusetts Institute of Technology, Cambridge, Mass., U.S.A.

The study of biology is partly an exercise in natural esthetics. We derive

much of our pleasure as biologists from the continuing realization of how

economical, elegant and intelligent are the accidents of evolution that have

been maintained by selection. A virologist is among the luckiest of biologists

because he can see into his chosen pet down to the details of all of its mole-

cules. The virologist sees how an extreme parasite functions using just the most

fundamental aspects of biological behavior.

A virus is a form of life with very simple requirements (1). The basic

needs of a virus are a nucleic acid to be transmitted from generation to gen-

eration (the genome) and a messenger RNA to direct the synthesis of viral

proteins. The critical viral proteins that the messenger RNA must encode are

those that coat the genome and those that help replicate the genome. One of

the great surprises of modern virology has been the discovery of the variety of

genetic systems that viruses have evolved to satisfy their needs. Among the

animal viruses, at least 6 totally different solutions to the basic requirements

of a virus have been found (2).

If we look back to virology books of 15 years ago, we find no appreciation

yet for the variety of viral genetic systems used by RNA viruses (3). Since

then, the various systems have come into focus, the last to be recognized

being that of the retroviruses (“RNA tumor viruses”). As each new genetic

system was discovered, it was often the identification of an RNA or a DNA

polymerase that could be responsible for the synthesis of virus-specific

nuclei acids that gave the most convincing evidence for the existence of the

new system.

Now that the life-styles of different types of viruses have been delineated

we can ask what relation there is between a virus’ multiplication cycle and

the disease it causes. In general, this question has no simple answer because

disease symptoms do not correlate with the biochemical class of the virus. For

instance, both poliovirus and rhinovirus are picornaviruses but one causes

an intestinal infection with paralysis while the other causes the common

cold. One class of RNA viruses, however, does have a unique symptom asso-

ciated with it: the biochemically-defined group of viruses called the retrovi-

ruses are the only RNA viruses known to cause cancer. For a virologist interest-

ed in cancer, the problem is to first understand the molecular biology of

retroviruses and then to understand how they cause the disease.

In what follows, I will review my personal involvement in uncovering the

different genetic systems of RNA viruses, a story which leads to the recogni-

tion of the unique style of retroviruses. I will then consider what is known

about the relationship between the biochemistry of retroviruses and their abil-

ity to be oncogenic.

As I tell my story I will mention a few of the many co-workers, teachers and

students who have influenced my thinking or contributed their labors and

ideas to the products of my laboratory. To mention all of the people to whom

I am indebted would make too long a list; I can only say that the honors I re-

ceive are in large measure testaments to their accomplishments.

P I C O R N A V I R U S E S

My work on the genetic systems of RNA viruses dates back to my graduate

school days. As part of my introduction to animal virology during a Cold

Spring Harbor course, I heard Richard Franklin describe his then-recent ex-

periments using autoradiography to show that Mengovirus, a picornavirus

and a close relative of poliovirus, could shut off the nuclear synthesis of cellu-

lar RNA early after infection and could later induce new RNA synthesis in

the cytoplasm which appeared to represent synthesis of viral RNA (4). I decid-

ed to go to the Rockefeller University as a graduate student with Richard

Franklin in order to work on the system he had developed.

Before I began to study how picornavirus RNA was made, it was already

known from the work of Simon that picornavirus RNA synthesis was inde-

pendent of DNA synthesis (5). Furthermore, studies with actinomycin D had

shown that neither synthesis nor expression of cellular DNA was involved in

viral RNA synthesis (6). These results suggested that Mengovirus might

make a cytoplasmic RNA-dependent RNA synthesis system. The concept that

viruses induce synthesis of their own enzymes had strong precedents in bacte-

riophage systems - Seymour Cohen’s work had shown decisively that new vi-

rus-specified enzymes were found in infected bacteria (7).

We approached the problem of the virus’ effect on intracellular RNA syn-

thesis as a question in enzymology. We first showed that the nuclei from

Mengovirus-infected cells were greatly reduced in their ability to carry out

cell-free DNA-dependent RNA synthesis compared to nuclei of uninfected

cells (8). Later, we showed that cytoplasmic extracts of Mengovirus - or

poliovirus-infected cells contained an RNA synthesis activity not evident in

uninfected cells and not inhibited by actinomycin D (9). When we learned