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Dr Richard C. Hunt

Medical Microbiology MBIM 650/720 Lectures 63 and 64

Cancers are the result of a disruption of the normal restraints on cellular proliferation. 
It is apparent that the number of ways in which such a disruption can occur is strictly 
limited and there may be as few as forty cellular genes in which disruption leads to 
unrestrained cell growth. There are two basic classes of these genes in which 
mutation can lead to loss of growth control: 
(a) Those genes that are stimulatory for growth and which cause cancer when 
hyperactive. Mutations in these genes will be dominant
(b) Those genes that inhibit cell growth and which cause cancer when they are 
turned off. Mutations in these genes will be recessive

Viruses are involved in cancers because they can either carry a copy of one of these 
genes or can alter expression of the cell's copy of one of these genes.



There are two classes of tumor viruses: the DNA tumor viruses and the 
RNA tumor viruses
, the latter also being referred to as RETROVIRUSES
We shall see that these two classes have very different ways of reproducing 
themselves but they often have one aspect of their life cycle in common: the ability to 
integrate their own genome into that of the host cell. Such integration is not, however, 
a pre-requisite for tumor formation.

If a virus takes up residence in a cell and alters the properties of that cell, the cell is 
said to be transformed.


Transformation often includes loss of growth control, ability to invade extracellular 
matrix and dedifferentiation. In carcinomas, many epithelial cells undergo an
epithelial-mesenchymal transformation. Transformed cells often exhibit chromosomal 

The region of the viral genome (DNA in DNA tumor-viruses or RNA in RNA-tumor 
viruses) that can cause a tumor is called an ONCOGENE. This foreign gene can be 
carried into a cell and cause it to take on new properties such as immortalization and anchorage-independent growth.

The discovery of viral oncogenes in retroviruses led to the finding that they are not 
unique to viruses and homologous genes (called proto-oncogenes) are found in  
cells. Indeed, it is likely that the virus picked up a cellular gene during its evolution 
and this gene has subsequently become altered. Normally, the cellular 
are not expressed in a quiescent cell since they are involved in 
growth (which is not occurring in most cells of the body) and development; or they are 
expressed at low levels. However, they may become aberrantly expressed when the 
cell is infected by tumor viruses that do not themselves carry a viral oncogene.  We 
shall see later how this happens but it is clear that a virus may cause cancer in two 
ways: It may carry an oncogene into a cell or it may activate a cellular proto-oncogene.

The discovery of cellular oncogenes opened the way to the elucidation of mechanisms
by which non-virally induced cancers may be caused. We shall investigate what the 
protein products of the viral and cellular oncogenes do in the infected cell and in cells 
in which cellular proto-oncogenes are expressed. We shall see that their functions 
strongly suggest mechanisms by which cells may be transformed to a 
neoplastic phenotype. The discovery of cellular oncogenes led to the discovery 
of another class of cellular genes, the tumor repressor (suppressor) genes or 

Initially, the involvement of viral and cellular oncogenes in tumors caused by 
retroviruses was much more apparent than the involvement of the DNA tumor virus 
oncogenes but the discovery of tumor repressor genes (as a result of our 
knowledge of how retroviruses cause cancer) led to the elucidation of the mode of 
action of DNA virus oncogenes.

It should be noted that while viruses have been vitally instrumental in elucidation of the 
mechanisms of oncogenesis, most human cancers are probably not the result of a 
retroviral infection although retroviruses are important in cancers in some animals.



The information flow in DNA tumor viruses is similar to that in eucaryotic cells

Figure 1

DNA tumor viruses have two life-styles:

In permissive cells, all parts of the viral genome are expressed. This leads to viral 
replication, cell lysis and cell death

In cells non-permissive for replication, viral DNA is integrated into the cell chromosomes
(usually but not always) at random sites. Only part of the viral genome is expressed. 
The early, control functions (e.g. T antigens) of the virus, are expressed. Viral structural 
proteins are not expressed and no progeny virus is released.



FAMILY: Papovaviridae - Papovaviruses


papilloma1.gif (289018 bytes) Papilloma virus Copyright 1994 Veterinary Sciences Division, Queens University Belfast  

papilloma2.gif (10784 bytes)  Papilloma virus Copyright Dr Linda M Stannard, 1995 (used with permission)

papilloma3.gif (15776 bytes) Papilloma virus Computer colorized EM image. All 72 capsomeres are pentamers of the major structural protein. Copyright Dr Linda M Stannard, 1995 (used with permission) 

Figure 2


Papilloma viruses are wart-causing viruses that also certainly cause human 
neoplasms and cause natural cancers in animals.

Warts are usually benign but can convert to malignant carcinomas. This occurs in 
patients with epidermodysplasia verruciformis (more here). Papilloma viruses 
are also found associated with human penile, uterine and cervical carcinomas and 
are very likely to be their cause; moreover, genital warts can convert to carcinomas.

epidermo.gif (48292 bytes)  Epidermodysplasia verruciformis. This widespread, markedly pruritic, erythematous eruption was eventually found to be caused by human papillomavirus infection. International Association of Physicians in AIDS Care  epidermo.jpg (15056 bytes) Verrucous carcinoma.  The epithelium shows surface maturation, parakeratosis, and hyperkeratosis. There is little or no cellular atypia. The stroma shows a mild chronic inflammatory infiltrate. The Johns Hopkins Autopsy Resource (JHAR) Image Archive. 

Figure 3


Squamous cell carcinomas of larynx, esophagus and lung  appear very like cervical 
carcinoma histologically and these may also involve papilloma viruses.

There are 51 types of papilloma viruses but, clearly, not all are associated with cancers; 
however, papillomas may cause 16% of female cancers worldwide and 10% of all 

Vulvar, penile and cervical cancers associated with type 16 and type 18  
papilloma viruses (and others) but the most common genital human papilloma viruses 
(HPV) are types 6 and 11. As might be expected if they are indeed the causes of 
certain cancers, types 16 and 18 cause transformation of human keratinocytes. In a 
German study, it was shown that 1 in 30 HPV type16-infected women will develop 
malignant disease while 1 in 500 infected people develop penile or vulvar cancer. 
Since not all infected persons develop a cancer, there are probably co-factors in 
stimulating the disease. Such co-factors have been identified in alimentary tract 
carcinomas in cattle where a diet containing bracken fern is associated with the 

Nevertheless, the epidemiological data are very strong.




sv40.jpg (50763 bytes) Transmission electron micrograph of polyomavirus SV40   Dr. Erskine Palmer  CDC

Figure 4

Simian virus 40
SV 40 is a monkey polyoma virus that causes sarcomas in juvenile hamsters. It was 
isolated from normal  monkey kidney cells in which it replicates.

Polyoma virus
Polyoma virus was so named because it causes a wide range of tumors in a number 
of animal species. It was originally isolated from AK mice and is fully permissive for 
replication in mouse cells. It  causes leukemias in mice and hamsters.

Human polyoma viruses
There are two human polyoma isolates, known as BK and JC; neither came from a 
tumor but they will cause tumors when injected into animals. 70-80% of the population 
is seropositive for JC. This virus causes progressive multifocal leukoencephalopathy 
(see section on slow virus diseases), a disease associated with immunosuppression. 
In 1979, the rate of occurrence of this disease was 1.5 per 10 million population. It has 
become much more common because of AIDS and is seen in 5% of AIDS patients.

Note: Polyoma viruses are usually lytic and when transformation occurs, it is because 
the transforming virus is defective. After integration into host DNA, only EARLY 
are transcribed into mRNA and expressed as a protein product. 
These are the TUMOR ANTIGENS. Because the expression of the genes for tumor 
antigens is essential for transformation of the cells, they may be classified as



SV 40 Tumor antigens are oncogenes

Large T antigen

Necessary for transformation of cell to cancerous state

Stimulates host cell to replicate its DNA

Found in nucleus and at cell surface (tumor-specific transplantation antigen)

Binds to cell DNA

Binds to p53 (see below)

In polyoma there is also middle T antigen which can also act as an oncogene.

Two important points about T antigens of DNA tumor viruses as 

1) They are true viral genes. There are no cellular homologues in the uninfected cell.

2) They are necessary in lytic infections because they participate in the control of viral
and cellular DNA transcription.

These properties should be contrasted  with retroviral oncogenes to be 
discussed later


FAMILY: Adenoviridae


adeno1.gif (36650 bytes) Adenovirus Copyright Dr Stephen Fuller, 1998 

adeno2.gif (35105 bytes)  Adenovirus  Copyright Dr Linda M Stannard, University of Cape Town, South Africa, 1995 (used with permission).

Figure 5

 These viruses are highly oncogenic in animals and only a portion of the virus is 
integrated into the  host genome. This portion codes for early functions (E1A region 
contains the oncogenes that code for several T antigens). No humans cancers have 
been unequivocally associated with adenoviruses. E1A gene product (early 
non-structural protein) binds to the product of Rb gene (see below). Thus polyoma 
and adenoviruses seem to cause cell transformation in a similar manner: the 
integration of early function genes into the chromosome and the expression of these 
DNA synthesis-controlling genes without the production of viral structural proteins.


FAMILY: Herpesviridae


herpes.gif (48070 bytes) Herpes virus. Negative stain Copyright Dr Linda M Stannard, University of Cape Town, South Africa, 1995 (used with permsssion).  herpes2.gif (9908 bytes)  Liquid-Crystalline, Phage-like Packing of Encapsidated DNA in Herpes Simplex Virus (F.P.Booy, W.W.Newcomb, B.L.Trus, J.C.Brown, T.S.Baker, and A.C.Steven, in CELL, Vol 64 pp 1007-1015, March 8, 1991)

herpes simplex.jpg (32705 bytes) Herpes Simplex Virus (TEM x169,920)  Copyright Dr Dennis Kunkel (used with permission)


Figure 6

There is considerable circumstantial evidence that implicates these enveloped 
DNA viruses in human neoplasms. They are highly tumorigenic in animals. It is 
notable that herpes viruses exist primarily as episomes in the cell and do not 
integrate into the host cell genome. By the time that tumors arise, no trace of the 
virus can usually be found. Herpes virus DNA is found in only a small number of 
herpes-transformed cells. They may have a hit and run mechanism of oncogenesis,
perhaps by causing chromosomal breakage or other damage. See below.

Epstein-Barr virus

This is the herpes virus that is most strongly associated with cancer.  It is causally 
associated with:     

Burkitt's lymphoma (but see below) in Africa 
Nasopharyngeal cancer in other areas (common in China and SE Asia)
B cell lymphomas in immune suppressed individuals (such as in organ transplantation or HIV)
Hodgkin's lymphoma. EBV has been detected in a high percentage of 
Hodgkin's lymphomas (found in about 40% of affected patients)

EBV can cause lymphoma in Marmosets and transform human B lymphocytes 
in vitro. 

EBV causes infectious mononucleosis. Why this virus causes a benign 
disease in some populations but malignant disease in others is unknown.

burkitta.jpg (19017 bytes)  Burkitt's Lymphoma The Johns Hopkins Autopsy Resource (JHAR) Image Archive. 

Figure 7

Human cytomegalovirus

This herpes virus is frequently associated with Kaposi's sarcoma but  this is now 
thought probably to be  caused by a newly-discovered herpes virus, 
human herpes virus 8

Herpes simplex II

This virus was associated in epidemiological studies with cervical cancer. Now the 
evidence for papilloma virus is better


FAMILY: Hepadnaviridae


hep2.gif (61292 bytes) Hepatitis B virions: two exposed cores (indicated by arrows) hep3.gif (87435 bytes) Hepatitis B virions hep4.gif (22924 bytes) A diagrammatic representation of the hepatitis B virion and the surface antigen components hepatitis.gif (36800 bytes)  All four images: Copyright Dr Linda M Stannard, University of Cape Town, South Africa, 1995 (used with permission). 

Figure  8


Hepatitis B virus is very different from the other DNA tumor viruses. Indeed, even 
though it is a DNA virus, it is much more similar to the oncornaviruses (RNA tumor 
viruses) in its mode of replication. Hepatitis B is a vast public health problem and  
hepatocellular carcinoma (HCC),  which is one of world's most common cancers, 
may well be caused by HBV. There is a very strong correlation between HBsAg 
(hepatitis B virus surface antigen) chronic carriers and the  incidence of HCC. 
In  Taiwan, it has been shown that HBsAg carriers have  a risk of HCC that is 217 times 
that of a non-carrier. 51% of deaths of HBsAg carriers are caused by liver cirrhosis or 
HCC compared to 2% of the general population.

hepato-b.jpg (82335 bytes)  This woman has hepatitis B and is suffering from liver cancer. She was a Cambodian refugee and died 4 months after she arrived in a refugee camp (average life expectancy after diagnosis of liver cancer is 6 months) Immunization Action Coalition Courtesy of Patricia Walker, MD, Ramsey Clinic Associates, St. Paul, MN

Figure 9

NOTE: Hepatitis B virus is a DNA tumor virus BUT it has a very weird way of 
replicating itself. The DNA is transcribed into RNA not only for the manufacture of 
viral proteins but for genome replication. Genomic RNA is transcribed back into 
genomic DNA. This is called REVERSE TRANSCRIPTION. This is not typical 
of DNA tumor viruses but reverse transcription is a very important factor in the 
life cycles of RNA-tumor viruses. See below.


Australian National University Hepadnaviridae information




HIV1.jpg (9840 bytes) Human immunodeficiency virus  Copyright Department of Microbiology, University of Otaga, New Zealand. Structure of a retrovirus: (The virus shown is human immunodeficiency virus-1)  From the Harvard AIDS Institute Library of Images, courtesy of Critical Path AIDS Project, Philadelphia.

Figure 10

Retroviruses are different from DNA tumor viruses in that their genome is RNA but 
they are similar to many DNA tumor viruses in that the genome is integrated into host 

Since RNA makes up the genome of the mature virus particle, it must be copied to 
DNA prior to integration into the host cell chromosome. This life style goes against 
the central dogma of molecular biology in which DNA is copied into RNA.

  Retrovirus replication

Figure 11

Retrovirus structure

The outer envelope comes from the host cell plasma membrane

Coat proteins (surface antigens) are encoded by env (envelope) gene. One primary 
gene product is made but this is cleaved so that there are more than one surface 
glycoprotein in the mature virus (cleavage is by host enzyme in the 
Golgi apparatus

Inside the  membrane is an icosahedral capsid containing proteins encoded by the 
gene (Group- specific AntiGen). Gag- encoded proteins also coat the genomic 
RNA. Again there is one primary gene product. This is cleaved by a 
proteins (from the pol gene)

There are two molecules of genomic RNA per virus particle with a 
5' cap and a 3' poly A sequence. Thus, the virus is diploid. The RNA is plus sense
 (same sense as mRNA).

About 10 copies of reverse transcriptase are present within the mature virus
these are encoded  by the pol gene.

Pol gene codes for several functions (again, as with gag and env, a polyprotein is 
made that is then cut up)

The pol gene products are:

a) Reverse transcriptase (a polymerase that copies RNA to DNA)

b) Integrase (integrates the viral genome into the host genome)

c) RNase H  (cleaves the RNA as the DNA is transcribed so that reverse transcriptase can make the second complementary strand of DNA)

d) Protease (cleaves the polyproteins translated from mRNAs from the gag gene and the pol gene itself). Note: this is a virally encoded protease and the target of a new generation of anti-viral drugs.

  Structure of RSV protease bound to a peptide analog of the HIV cleavage site
Requires Netscape and a Chime plug-in. Get Chime 
- Click on thumbnail to open file

Figure 12



These are the  tumor viruses and those with similar morphology. The first member of 
this group to be discovered was Rous sarcoma virus (RSV)- which causes a slow 
neoplasm in chickens.

htlv-pic.jpg (53579 bytes)  Human T-lymphocyte Virus Attacking a T-lymphocyte (TEM x26,400)  Copyright Dr Dennis Kunkel (used with permission)

Figure 13

Viruses in this group that cause  tumors in humans are: 

HTLV-1 (human T-cell lymphotropic virus) (Information Box): Causes Adult T-cell 
leukemia (Sezary T-cell leukemia) which is found in some Japanese islands, 
the Caribbean, Latin America (Information Box)  and  Africa.  HTLV-1 is sexually 
transmitted  (Information Box) 

HTLV-2: Hairy cell leukemia (Information Box)

These have a long latent period; they are mainly associated with diseases of 
ungulates (e.g. visna virus) but HIV (formerly HTLV-III) which causes AIDS belongs 
to this group. It is much more closely related to some Lentivirinae than it is to HTLV-I 
and HTLV-II which are Oncovirinae

There is no evidence of pathological effects of these viruses.



hivstage.gif (28491 bytes)  Stages in the productive infection of a cell by a retrovirus

Figure 14

The following stages occur in the infection process:

1) Binding to a specific cell surface receptor

2) Uptake by endocytosis or by direct fusion to the plasma membrane. The virus 
may need entry into a low pH endosome  before fusion can occur although some 
(e.g. HIV) can fuse directly with the plasma membrane 

3) RNA (plus sense) is copied by reverse transcriptase to minus sense DNA. Here, 
the polymerase is acting as an RNA-dependent DNA polymerase. Note: reverse
transcriptase is a DNA polymerase and it therefore needs a primer. This is a tRNA 
that is incorporated into the virus particle.

4) RNA is displaced and degraded by a virus-encoded RNase H activity. Reverse 
transcriptase now acts as a DNA-dependent DNA polymerase and copies the 
new DNA into a double strand DNA. This is the provirus.

5) Double strand DNA is integrated into host cell DNA (see below) using a virally 
encoded integrase enzyme. This DNA is copied every time cellular DNA is copied. 
Thus, at this stage the provirus is just like a normal cellular gene.

6) Full length, genomic RNA (plus sense) is copied from integrated DNA by 
host RNA polymerase II
. It is capped and poly adenylated

Since the full length genomic RNA is the same sense as message, it also acts as the mRNA for GAG and POL polyproteins.

The genomic RNA is spliced by host nuclear enzymes to give mRNA for other proteins such as ENV. The RNA of some more complex retroviruses such as HTLV-1 and HIV undergoes multiple splicing (see HIV notes).

Note: mRNA comes from splicing genomic RNA or is the genomic RNA. As a 
result, both mRNA and genomic RNA must be the  same sense - since mRNA 
must be plus sense, the genomic RNA of all retroviruses must also be plus sense.

An advantage of this mode of replication is that it allows growth in terminally 
differentiated cells since the only host cell polymerase usurped by the virus is 
RNA polymerase II which is present in all cells.



 If host RNA polymerase II is used to copy the DNA back to RNA, there are major 
problems with having a DNA provirus form but an RNA genome in the mature virus 

These problems include: 

1) RNA polymerase II does not copy the upstream and down stream control sequences of genes. It only copies the information necessary to make a 

2) The lack of proof reading by RNA polymerase II

Failure to copy the entire gene
The problem is that, when transcribing genes, RNA polymerase II needs control and 
recognition  sites upstream from the transcription initiation site. The upstream 
site at which the polymerase molecule binds is called the PROMOTOR. Promotors 
are not themselves copied into mRNA since they have no function in the translation 
of protein. After binding to the promotor, the polymerase begins transcription at a 
downstream site, the RNA initiation site. The polymerase continues to transcribe 
DNA into RNA until it reaches a termination/polyadenylation signal, part of which 
is not copied since it also has no function in the making of the protein. Furthermore, 
both up and downstream from the transcribed region are control sequences that 
modulate the transcription of the gene. These are called ENHANCERS
These are essential parts of any gene and must be present for RNA polymerase II 
to work but they are not copied to RNA.
This is because RNA polymerase II in the 
host cell has the function of making messenger RNA which is dispensed with after 
translation. To make a protein, the actual mRNA molecule does not need the controls
equences of the original gene. Thus, the use of host RNA polymerase II means 
that the control sequences in the original genome will not get into the RNA genome 
of progeny virions.

This means that either the DNA copy of the viral RNA genome virus must integrate 
into host DNA downstream from a host promotor and upstream from host 
termination sites
(a tall order indeed!) or it must find a way of providing its own 
control sequences (which, as we said, are not copied into progeny genome). It does 
the latter in a most complex manner.


How can a retrovirus provide its own control promotors and enhancers if 
they are not transcribed when the DNA provirus is copied to the genomic 
RNA form?

A rnacr.gif (3677 bytes)  The structure of the RNA genome of the mature retrovirus B dnacr.gif (4107 bytes) The genome structure of the DNA proviral form of a retrovirus

Figure 15

Here is a brief (and very incomplete) summary of how a retrovirus does it:

1) The viral RNA is composed of three regions. At each end are repeats (called, not 
surprisingly, terminal repeats). The repeat sequences (R) (shown in green) do not 
code for proteins. In between the two repeats, there is a unique (not repeated) region 
that contains the viral genes that code for the proteins (GAG, POL and ENV) plus other
unique sequences at either end that do not code for protein. At the 5' end of the RNA 
genome is the U5 region and at the 3' end is the U3 region. PBS (in above diagram) 
is the primer binding site. The tRNA binds here when RT starts copying. PPT is a 
polypurine tract.

2) In the integrated form (when transcribed into DNA and inserted into the host cell chromosome), the provirus is more complicated. We find that part of the 3' unique 
region (called U3) of the RNA genome has been copied and transposed to the 
opposite end of the genome. Conversely, part of the 5' end of the unique region 
(called U5) has been copied and transposed to the other end. This gives the 
integrated DNA the structure shown in figure 15B.

For convenience, only one strand of the DNA is shown. Now, of course, there are 
larger terminal repeats since the U3 and U5 regions are also repeated. 
The U3-R-U5 regions are known as long terminal repeats or LTRs
The U3 region contains all of the promotor information that is necessary to start 
RNA transcription at the beginning of the R (repeat) region while the U5 region 
contains all of the information necessary to terminate after the other R repeat. 
In addition, the LTRs contain information that enhances the degree of transcription of 
the three retroviral genes (enhancer regions). These enhancers can be up or 
downstream from the protein-encoding part of  the  genes.

Shockwave movie of LTR formation (go here) Requires Shockwave plug-in

Host RNA polymerase II copies the provirus DNA to genomic RNA which can be 
also spliced to mRNAs. Since the polymerase starts after the promotor (in U3), at 
the transcription initiation site, it begins exactly at the beginning of the R region (see 
diagram below). Thus we get a faithful (almost-see below) copy of the RNA that 
entered the cell. The termination sequences and poly A signal are in U5 which is 
also not copied.

ltr3cr.gif (4128 bytes)   The transcription of a retrorviral DNA with LTRs by RNA polymerase II results in the loss of the LTRs

Figure 16                Animated version here (requires IE5)

Note: Because of this mechanism, there can be only one promotor site (from U3) 
for all three viral genes so they must be all transcribed together. Splicing 
(enzymes from the host cell nuclear splicing machinery) gives the individual mRNAs 
where necessary. See notes on HIV in which this has been well elucidated. Unlike the 
situation with DNA tumor viruses, there is no distinction between early/late functions.

Note: You may ask why, if U5 contains the termination and polyadenylation sites, 
does the transcript not just terminate after the first R region in the above diagram and 
never get into the structural genes. The termination site in the first U5 is suppressed, 
often by complex secondary structure mechanism. In some retroviruses there is a 
sequence in the gag gene that provides the context to suppress the termination 
activity of the first U5. Clearly the second U5 does not have a gag gene following it.

Note: The copying of the RNA and the synthesis of the complementary DNA strand 
are carried out by reverse transcriptase. Reverse transcriptase is an 
RNA-dependent DNA polymerase and, like all DNA polymerases, it needs a  
. This is a cellular tRNA that is packaged within the viral particle.

This strategy of virus replication in which viral RNA is first copied to DNA (by reverse transcriptase) which then gives rise to mRNA and protein poses a problem for the 
virus. The initial step (RNA to DNA) is carried out by a viral enzyme which is not 
normally in the cell. Yet this transcription step must occur before any mRNA 
transcription or protein translation can occur. The problem is solved by the virus 
carrying about 10 copies of the reverse transcriptase protein into the cell with it. 
These were packaged when the virus was assembled in the previous host cell.



onco2.jpg (57386 bytes) Typical retrovirus structure and the structure of a retrovirus with an oncogene (Rous Sarcoma Virus)

Figure 17

The structure shown in figure 15A and the upper part of figure 17 is that of a typical 
retrovirus with three structural genes (gag, pol and env) but none of these is 
 If the virus is to transform a cell, in addition to, or instead, of part of the gag/pol/env 
genome, it must have sequences that alter cellular DNA synthesis and provide the 
other functions that are typical of a transformed cell. Thus we also find an 
in the viral genome of many retroviruses that transform cells to 
neoplasia (figure 17).

Definition of virally-induced transformation: The changes in the biologic 
function and antigenic specificity of a cell that result from integration of viral genetic 
sequences into the cellular genome and that confer on the infected cell certain 
properties of neoplasia. Note, however, that transformation can be induced by 
factors other than viruses e.g. carcinogens.


In retroviruses, these were first discovered as an extra gene in Rous sarcoma 
(RSV). This gene was called src (for sarcoma). 
src is not needed for viral replication
. It is an extra gene to those (gag/pol/env) 
necessary for the continued reproduction of the virus. RSV has a complete 
gag/pol/env genome. Deletions/mutations in src abolish transformation and 
tumor promotion but the virus is still capable of other functions. RSV is unusual in that 
it has managed to retain its whole genome of gag/pol/env. 

onco3.jpg (90987 bytes) In sharp contrast to RSV, many retroviruses have lost part of 
their genome to accommodate an oncogene. This has two consequences:

1) The protein encoded by the oncogene is often part of a fusion protein with other 
virally-encoded amino acids attached

2) Virus is in trouble as cannot make all of itself. To replicate and bud from the host 
cell needs products of another virus, that is a helper virus

About forty oncogenes have now been identified. Note that they are referred so by a 
three letter code (e.g. src, myc) often reflecting the virus from which they were first 
isolated. Some viruses can have more than one oncogene (e.g. erbA, erbB). Here 
are a few of the most studied:



Rous sarcoma virus  v-src
Simian sarcoma virus v-sis
Avian erythroblastosis virus v-erbA or v-erbB
Kirsten murine sarcoma virus v-kras
Moloney murine sarcoma virus v-mos
MC29 avian myelocytoma virus v-myc



Once retroviral oncogenes had been discovered, a surprising observation was 
made: Unlike the situation with DNA virus oncogenes which are true viral genes
there are homologs of all retrovirus oncogenes in cells that are not infected by a 
retrovirus. These cellular homologs are often genes involved in growth control and development/differentiation (as might be expected) and have important 
non-transforming functions in the cell; some can cause cancer under certain 
circumstances and, presumably, those not shown to cause cancer have the ability to 
do so under the correct conditions. The cellular homologs of viral oncogenes are 
called proto-oncogenes. To distinguish viral oncogenes from 
cellular proto-oncogenes, they are often referred to as v-onc and c-onc respectively. 
Note: c-oncs are not identical to their corresponding v-oncs. It appears that the virus 
has picked up a cellular growth controlling or differentiation  gene and, after the gene 
was acquired by the virus, it has been subject to mutation.

Definition of a proto-oncogene: A host gene that is homologous to an oncogene 
that is found in a virus but which can induce transformation only after being altered 
(such as mutation or a change of context such as coming under the control of a highly 
active promotor). It usually encodes a protein that functions in DNA replication or 
growth control at some stage of the normal development of the organism.



1) These are typical cellular genes with typical control sequences. As with most 
eucaryotic genes, most have introns (retroviral oncogenes do not)

2) They show normal Mendelian inheritance

3) As with all genes in the eucaryotic genome, they are always at same place in 
genome (cf. what would be expected of endogenous retroviruses that had, over time, 
become incorporated into the cellular genome)

4) There are no LTR sequences (v-oncs always are in an LTR context)

5) Viral oncogenes are most like the c-onc of the animal from which the virus is thought 
to have acquired the gene. Thus, v-src of RSV is more like chicken src than human 
src. Note: v-onc was picked up accidentally by the virus from the genome of a 
previous host cell

6) Cellular oncogenes are expressed by the cell at some period in the life of the cell, 
often when the cell is growing, replicating and differentiating normally. They are 
usually proteins that are involved in growth control.

7) Cellular oncogenes are highly conserved


If v-onc and c-onc are so alike, why does the viral oncogene (v-onc) introduced by a 
virus cause havoc in the cell? This is due to differences in the genes, mutations that 
have occurred in the gene once it was picked up by the virus. Such changes include:

1) Amino acid substitutions or deletions--gives different translation products

2) Many v-oncs are fusion proteins

3) V-oncs are  inserted into the host genome along with LTRs which contain promotors/enhancers. This is likely to result in over expression of a gene that we 
know  is probably involved in control of DNA transcription and replication!



The observation that an acutely transforming virus such as RSV contains an extra 
gene, the oncogene, explains their high neoplastic potential but, in contrast, 
chronically transforming
retroviruses only produce tumors  slowly and they carry 
gene equivalent to a v-onc. At best, these viruses have just the three usual 
viral genes (gag/pol/env). An example is avian leukosis virus (ALV).

How do chronically transforming viruses induce a tumor if they do not have an 

A seminal observation was made: Just as any other retrovirus does, ALV can 
integrate into the cell genome at many different sites but, in ALV-induced tumors
the virus is ALWAYS found in a similar position (very important!). This means that 
the crucial transforming event must be rare and that the cells that form the tumor are 
a clone (cf. the acute transformers which integrate all over the place). In all cases of 
ALV-induced tumors, the viral genome is inserted near a cellular gene called 
. This is the cellular proto-oncogene that, in an altered form (i.e. as a v-onc), 
is carried by some acutely transforming retroviruses (e.g. avian 
myelocytoma virus
which causes carcinoma, sarcomas and leukemias). 
In addition, the level of translation of c-myc in the ALV-transformed cell is much 
greater than in uninfected cells. Thus, inserting the genome of ALV and other 
chronically transforming retroviruses next to a c-onc has the same effect 
as carrying in a v-onc.

proins2.jpg (68584 bytes) So, in integration, the virus comes to lie upstream from c-myc which 
then comes under the influence of the strong LTR promotors of the virus which leads 
to over expression of c-myc. This is called oncogenesis by promotor insertion

enhanins2.jpg (82601 bytes) But in some tumors the virus is downstream from the c-myc gene. 
However, we saw that LTRs also have enhancers in addition to promotors. We 
know that enhancer sequences can be upstream or downstream to have their effect. 
This is called oncogenesis by enhancer insertion

Why is insertion near c-myc important? The protein coded for by this gene is found in 
the nucleus of normal cells and is involved in control of DNA synthesis. It can be 
shown that over-expression of c-myc leads to rapid DNA replication.



Once it had been shown that viruses can either bring an oncogene into the cell or can 
take control of a cellular proto-oncogene to give rise to a tumor, the question arose of
 whether cellular proto-oncogenes could give rise to tumors in the absence of 
retroviral infection. The answer is yes! Other chromosomal rearrangements can bring 
a c-onc under the control of the wrong promotor/enhancer. Alternatively, the c-onc 
might be mutated in a particular way so that it was over-expressed or it might code 
for a mutant protein with an altered function.

chromo.jpg (83345 bytes) Chromosomal mapping allows the precise localization of the site 
of a gene on a particular chromosome and many cancers are associated with 
alterations in chromosomes, particularly translocations (the breakage of a 
chromosome so that the two parts associate with two parts of another chromosome). 

chromo2.jpg (107857 bytes) Many break sites in tumor cells are very close to a known c-onc. 
This is highly suggestive and unlikely to have occurred by chance!




Burkitt's lymphoma *


8 to 14

Acute myeloblastic leukemia


8 to 21

Chronic myelogenous leukemia


9 to 22

Acute promyelocytic leukemia


15 to 17

Acute lymphocytic leukemia


6 deletion

Ovarian cancer


6 to 14

* In Burkitt's lymphoma the c-myc on chromosome 8 is brought to a site on 
chromosome 14 close to the gene for immunoglobulin heavy chains
It seems that the proto-oncogene may thus be brought under the control of the 
Ig promotor, which is presumably very active in B lymphocytes. This explains why 
this tumor arises in B cells. In other lymphomas, a c-onc is brought next to the 
immunoglobulin light chain promotor. These are also B cell lymphomas.

Epstein-Barr virus is probably the cause of Burkitt=s lymphoma. This is a 
herpes virus and herpes viruses commonly cause chromosomal breaks.



The best evidence comes from the cellular oncogene that is the homologue of the 
viral oncogene found in the Harvey strain of murine sarcoma virus (the v-onc is called
). This c-onc was isolated from bladder carcinomas and compared to the 
normal c-onc proto-oncogene. In many tumor cells only one change was found in the 
amino acid sequence of the protein, glycine at amino acid position 12 was changed 
to valine. At position 12 only glycine and proline gave normal growth. All other amino 
acids at this position gave a transformed cell. In a lung carcinoma, the 
transforming DNA also contained c-HaRas, again it had a point mutation, this time at 
position 61.



As mentioned above, c-oncs are normal cellular genes that are expressed and 
function at some stage of the life of the cell.
We should expect them to be involved 
in DNA synthesis or perhaps the signaling pathways that lead to proliferation. 
More than 40 oncogenes have been identified and there are probably a few 
undiscovered ones.

We can sub-divide the cellular oncogenes into those that encode nuclear proteins 
and those that encode extra-nuclear proteins. The latter are mostly associated with 
the plasma membrane of the cell.

Products of oncogenes that are nuclear proteins: e.g. myc, myb. These are 
involved in control of gene expression (that is the regulation of transcription-they are 
transcription factors) or the control of DNA replication. Neoplasia is associated with 
elevated transcription of the oncogene but strong expression is not always necessary,
rather there is a need to make the gene constitutively active rather than under control 
of normal regulatory processes.

Products of oncogenes that are cytoplasmic or membrane-associated 
: e.g. abl, src, ras. This type does not exhibit altered expression but seems 
to convert from proto-oncogene to oncogene by mutation. Thus, in src-induced tumors,
strong over expression of the oncogene has no effect.



Control of DNA transcription (found in nucleus) myc
Signaling of hormone/growth factor binding such as a tyrosine kinase src is a membrane-bound tyr kinase.
GTP-binding proteins involved in signal transduction from a surface receptor to the nucleus ras
Growth factors sis is an altered form of platelet-derived growth factor B chain
Growth factor receptors erb-B is a homolog of the epidermal growth factor receptor (it is also a tyrosine kinase). fms is a homolog of the macrophage colony-stimulating factor (M-CSF) receptor

 onco1.jpg (78886 bytes) Ways in which altered proto-oncogenes might lead to 
cell transformation


 Classes of cellular proto-oncogene products 


GF = growth factors
REC = membrane receptors
GP = G-protein transducers of signals
KINASE = membrane bound tyrosine kinase
CYT KINASE = cytoplasmic protein kinase


In each of these cases, the mutation is dominant. Thus, for example if one 
allele of erb-B (a homolog of the EGF receptor) is mutated so that it is a constitutively 
switched on (i.e does not need epidermal growth factor to bind to switch on the 
tyrosine kinase activity), then the signal is on, regardless of the fact that the other 
allele is normal.


ANTI-ONCOGENES (Tumor suppressor genes)

The way in which retroviruses cause tumor formation via oncogenes was established
before anything was known about how DNA tumor viruses cause tumors. Certainly, 
DNA tumor viruses carry oncogenes (e.g. SV40 T-antigen) but how do these proteins, 
encoded in true viral genes with no cellular homologs
, cause the formation 
of tumors?

It has long been known that most tumors are the result of dominant mutations, i.e. 
a function is gained that makes the cell grow when it should not. For example, 
as noted above, if we have a receptor that sends a signal when it binds a growth 
factor by switching on its tyrosine kinase activity and that receptor becomes mutated 
so that its tyrosine kinase activity is permanently activated, the cell will get the 
aberrant growth signal even in the heterozygote. Thus the mutant allele is dominant 
over the normal allele.

 protonc.jpg (94362 bytes)    Dominant mutations are function gained 

Retinoblastoma: A recessive tumor

There is a curious class of tumors that do not fit the usual characteristics in which the 
mutant oncogene is dominant over the wild type.

In retinoblastoma, there appears to be a lesion that is recessive, that is the cancer 
causing mutation causes a loss of function. (This is recessive because, in a 
diploid organism, there are two genes. If one allele is mutated so that it does not 
work, the other can still code for the normal protein and function is retained. 
In order to lose the function and have no protein being made, both genes must be 
mutated, i.e. we have a recessive mutation)
. Thus it appears that the protein that is 
encoded in the retinoblastoma (Rb) gene is a growth suppressor. If a 
mutation occurs in the Rb gene, there will be no Rb gene product at all
and the cell will grow abnormally because the growth suppressor is no longer present.
 The product of the Rb gene has been identified and shown to be a nucleus-located 
protein of 105 kDaltons.

antionc.jpg (101963 bytes)  Recessive mutations are functions lost 

A heterozygote at the Rb allele still has normal Rb and tumors can still be 
suppressed but homozygote has no functional Rb and tumors cannot be suppressed

 Above, we have noted that the adenovirus E1A (early function) protein is somehow 
involved in tumorigenesis. It has been found that E1A protein in the transformed adenovirus-infected cell is complexed with a 105kD protein! This turns out to be the 
Rb gene product. Thus, it seems that adenovirus may cause a cell to grow 
abnormally by complexing (and thereby inactivating) a cellular protein whose normal 
function is growth inhibition. Tumors caused by the inactivation of Rb gene product 
are, however, rather rare.

rbadeno.jpg (85811 bytes) Rb and adenovirus E1A 

 p53 and human cancer

Over the past two decades, since its discovery in 1979, a gene known as the 
p53 gene (after the size of its encoded protein) has been linked to many cancers 
including many that are inherited. In these inherited cancers, it turns out that the 
p53 gene is mutant. Alterations in this protein seem to be the basis (direct or indirect)
 of most human cancers.
In total, 60% of human cancers involve p53

Human cancers that involve p53

cervix liver
breast lung
bladder skin
prostate colon

80% of colon cancers involve the p53 gene

Initially, it was thought that the p53 gene product caused cancers but further 
investigation showed the opposite, p53 is, like the retinoblastoma gene product, 
a tumor suppressor
. p53 protein has been referred to as The Guardian of the 
since it regulates multiple components of the DNA damage control system.

How does p53 work in a functional cell? Normally, there are only a few of the 
suppressor p53 molecules in a healthy cell and these are constantly turning over; 
but when the DNA becomes damaged (perhaps by radiation or chemical mutagens) 
and DNA replication results, p53 turnover ceases. The rise in p53 stops 
DNA replication.

p53 is a transcription factor. When it builds up, p53 binds to a specific site(s) on the 
chromosomes and switches on other genes and these, in turn, shut down mitosis. 
p53 can also act in another way, when it builds up it can set the cell on course to 
. Whether or not p53 causes reversible growth arrest or apoptosis depends
 on the state of cellular activation; for example, extensive, unrepaired DNA damage 
can lead to sustained p53 production committing the cell to apoptosis. In inherited 
cancers, there is a mutation in the p53 gene; often it is a single point mutation and the 
protein can no longer bind to its correct site on the DNA and so cannot suppress DNA replication.

Like the Rb gene, product, you would expect the effects of p53 to be recessive  
since the second normal p53 allele should make functional protein and should 
shut off DNA replication as usual; that is, if you are heterozygous for the mutation 
-- although, of course, you will only be one mutation away from  carcinogenesis
So why do cells that are heterozygous for the p53 mutation also have problems? 
Unfortunately, p53 protein forms tetramers in a ribbon-like array and so if half of the
 p53 proteins are mutant, there is a good chance that each tetramer will have one 
mutant p53 molecule and this inactivates the tetramer, a dominant-negative effect.

Although we have learned a lot from families that inherit p53 mutations, it is clear that 
most p53 mutations come from non-inherited environmental factors: carcinogens 
(benzopyrene in smoke, aflatoxin in molds on peanuts and corn, UV light) that result 
in point mutations. Note: there are also gain of function p53 mutations that lead to very
aggressive tumors. These turn on DNA replication genes.

What has this got to do with DNA tumor viruses? Just as with retinoblastoma gene 
product, the presence of a virus can mimic mutation and take the tumor suppressor 
out of action by complexing it in an inactive form that cannot bind to the specific site on
 DNA. This is what appears to happen in hepatitis C which causes hepatocellular
. In the case of a human papilloma virus-infected cell, p53 is 
directed to a protease that recognizes a cleavage site in p53, thereby destroying it.

Slide59.JPG (45450 bytes) p53, hepatitis C and papilloma virus 

Note: In radiation therapy, it was once thought that radiation damaged the DNA of 
dividing cells so that they could no longer divide. But in fact, the radiation only 
damages the DNA a bit which would not kill the cell but the bit of damage is enough to
up-regulate the production of p53. Much research is now going on to see whether one
 can introduce healthy p53 genes into cells to shut down tumor growth.

Thus, our knowledge of how retroviruses cause cancer has led to an understanding of the formerly cryptic manner in which DNA tumor viruses 
do the same thing.




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