1. Tumor Viruses Genome all viral proteins Replication Lysis Progeny virions Lytic Life Cycle For most viruses: www.freelivedoctor.com
2. Tumor Viruses Virus Cell Integration (often) Transformation Latent Life Cycle Some virus-specific proteins expressed (early functions) - No mature virus Viral structural proteins are not expressed Changes in the properties of host cell - TRANSFORMATION Sometimes latency may terminate – cell must be infected by complete virus www.freelivedoctor.com
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4. TRANSFORMATION VIRAL TRANSFORMATION The changes in the biological functions of a cell that result from REGULATION of the cell’s metabolism by viral genes and that confer on the infected cell certain properties characteristic of NEOPLASIA Tumor Viruses www.freelivedoctor.com
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6. Similar to host cell! www.freelivedoctor.com Two Major Classes of Tumor Viruses DNA Tumor Viruses DNA viral genome Host RNA polymerase Viral mRNA Viral protein DNA-dependent DNA polymerase (Host or viral)
7. Important: Use HOST RNA polymerase to make its genome An enzyme that normally makes mRNA IMPORTANT www.freelivedoctor.com RNA Tumor Viruses Viral RNA genome Reverse transcriptase (Virus-encoded) Viral DNA genome (integrated) DNA-dependent RNA polymerase ( Host RNA pol II) Viral genomic RNA Splicing (Host splicing enzymes) messenger RNA viral protein Virus
8. DNA Tumor Viruses DNA genome mRNA protein virus Host RNA polymerase II Host enzymes OR TRANSFORMATION In transformation usually only EARLY functions are expressed www.freelivedoctor.com
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11. DNA Tumor Viruses In Human Cancer Epidermodysplasia verruciformis Papilloma virus www.freelivedoctor.com
12. DNA Tumor Viruses In Human Cancer Squamous cell carcinoma: Larynx Esophagus All histologically similar Lung 10% of human cancers may be HPV-linked www.freelivedoctor.com Papilloma Viruses urogenital cancer wart malignant squamous cell carcinoma Papilloma viruses are found in 91% of women with cervical cancer
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16. DNA Tumor Viruses In Human Cancer Adenoviruses Highly oncogenic in animals Only part of virus integrated Always the same part Early functions E1A region: 2 T antigens E1B region: 1 T antigen E1A and E1B = Oncogenes www.freelivedoctor.com
17. DNA Tumor Viruses In Human Cancer ONCOGENE A gene that codes for a protein that potentially can transform a normal cell into a malignant cell An oncogene may be transmitted by a virus in which case it is known as a VIRAL ONCOGENE v-onc www.freelivedoctor.com
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21. DNA Tumor Viruses In Human Cancer Host enzyme Viral enzyme www.freelivedoctor.com Hepatitis B Virus DNA genome RNA polymerase II RNA Provirus Reverse transcriptase DNA genome
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25. RNA Tumor Viruses RNA Genome - Retroviruses RNA-dependent DNA Polymerase encoded by virus REVERSE TRANSCRIPTASE RNA genome Reverse transcriptase DNA genome Integrase Integrates Host RNA polymerase II RNA genome host www.freelivedoctor.com virus virus
34. RNA Tumor Viruses Bind to surface receptor www.freelivedoctor.com Retrovirus Life Cycle Endocytosis Fusion of membranes Release of nucleocapsid to cytoplasm Nucleus
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36. RNA Tumor Viruses Drawback to this lifestyle Genomic RNA DNA Genomic RNA Host RNA pol II Reverse transcriptase Pol II is a host enzyme that, in the uninfected cell, makes mRNA When making mRNA, pol II does not copy entire gene to RNA www.freelivedoctor.com
37. primer Viral genomicRNA Reverse transcriptase dsDNA Result: New copy of viral RNA is shorter - lacks control sequences Problem of using RNA pol II to copy a gene RT www.freelivedoctor.com promotor RNA synthesis initiation site RNA pol II RNA synthesis termination site
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39. RNA Tumor Viruses Clue: Difference in the two forms RNA R U5 GAG POL ENV U3 R www.freelivedoctor.com LTR Repeat region Repeat region DNA U3 R U5 GAG POL ENV U3 R U5 LTR
40. Viral RNA Reverse transcriptase R U5 U3 R U3 R U5 U3 R U5 Long terminal repeats are formed POLII www.freelivedoctor.com promotor RNA initiation site RNA termination site POLII
41. Retroviruses can have only one promotor LTR LTR U5 Therefore only one long RNA can be made Therefore mRNA requires processing Explains why RNA has to be positive sense POLII Contained in U3 www.freelivedoctor.com RNA initiation site RNA termination site POLII
42. R U5 GAG POL ENV U3 R Some retroviruses have an extra gene “ typical retrovirus” SRC www.freelivedoctor.com Rous Sarcoma Virus R U5 GAG POL ENV U3 R
43. Feline Sarcoma Virus (FSV) R U5 dGAG FMS dENV U3 R Avian Myelocytoma Virus (MC29) R U5 dGAG MYC dENV U3 R Avian Myeloblastosis Virus R U5 GAG POL MYB U3 R Some retroviruses have an oncogene instead of their regular genes www.freelivedoctor.com
46. RNA Tumor Viruses The discovery of the acutely transforming retroviruses that contain v-oncs explains how cancers may arise as a result of infection These viruses cause rapid cancer in animals in the laboratory www.freelivedoctor.com
47. RNA Tumor Viruses In contrast: Chronically transforming retroviruses cause tumors inefficiently after prolonged period of time No oncogene! – How does it cause a tumor? www.freelivedoctor.com R U5 GAG POL ENV U3 R Avian Leukosis Virus (causes lymphomas)
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49. RNA Tumor Viruses What is special about this site? Myelocytoma tumors from several birds all have the oncogene close to this site It is close to C-myc! Oncogenesis by promotor insertion www.freelivedoctor.com
50. RNA Tumor Viruses Could C-oncs be involved in NON-VIRAL cancers? www.freelivedoctor.com
51. RNA Tumor Viruses What do oncogenes encode? Proteins that are involved in growth control and differentiation Growth factors Growth factor receptors Signal transduction proteins Transcription factors www.freelivedoctor.com
52. DNA Tumor Viruses Herpes Genes can be assigned to sites on specific chromosomes mos and myc : chromosome 8 fes: chromosome 15 www.freelivedoctor.com fes mos myc myb
53. Cancers often result from gene translocations Burkitt’s Lymphoma 8:14 translocation Break in chromosome 14 at q32 Acute myelocytic leukemia 7:15 9:18 11:15:17 myc www.freelivedoctor.com
54. Oncogenesis by rearrangement Tumor c-onc new promotor Burkitt’s lymphoma myc (8) Ig heavy (8 to 14) Ig light (8 to 2) B-cell chronic lymphocytic bcl-1 Ig heavy (11 to 14) leukemia bcl-2 Ig heavy (18 to 14) T cell chronic lymphocytic tcl-1 T cell receptor leukemia (14 inversion) T cell chronic lymphocytic myc T cell receptor (8 to 14) leukemia www.freelivedoctor.com
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56. Proto-oncogenes Heterozygote Homozygote Allele 1 Allele 2 Allele 1 Allele 2 Normal Mutant Mutant Mutant Function gained Function gained Dominant mutations Binds under special circumstances Mutant always binds Mutant always binds Mutant always binds www.freelivedoctor.com Always binds Always binds
57. Anti-Oncogenes Rb Gene Mutant Rb Mutant Rb Rb Rb Rb protein Binds and controls cell cycle Turns off DNA replication No binding - Growth continues Mutant Rb Recessive mutations Function lost Heterozygote Homozygote www.freelivedoctor.com Mutation growth
58. Anti-Oncogenes Retinoblastoma gene has normal regulatory function in many cells Involved in Retinoblastoma Lung carcinomas Breast carcinomas www.freelivedoctor.com
In previous lectures we have looked at the replication of viruses. How the genome gives rise to viral proteins
Some viruses may lay latent in the cell for a very long time (and then may possibly lyse the cell). In this case only early functions of these viruses may be expressed. This results in virus proteins being expressed but no mature virus particles. The expression of these early virus proteins may nevertheless alter the properties of the infected cell. THE CELL IS SAID TO BE VIRALLY TRANSFORMED.
Viral genes interfere with cell replication control mechanism. NOTE: The early functions of DNA viruses do not make structural proteins but control cellular and viral genome replication.
Permissive cells: Replication, lysis and death Non-permissive cells: transformation. Usually DNA is integrated. Early functions only are expressed. Control information, rather than structural proteins
Squamous cell carcinomas of larynx, espophagus and lung are all histologically similar to cervical carcinomas. HPV also linked to penile and vulval cancers 10% of all human cancers and 16% of all female cancers may be HPV-linked
Squamous cell carcinomas of larynx, espophagus and lung are all histologically similar to cervical carcinomas. HPV also linked to penile and vulval cancers 10% of all human cancers and 16% of all female cancers may be HPV-linked
No final proof that these viruses cause cancer as Koch’s postulates cannot be fulfilled. Papilloma viruses cannot be grown in culture It appears that there is free plasmid in the cell rather than integration
SV40 was in early batches of polio vaccine Normally these viruses cause lytic infection and transform when they are incomplete
Adenovirus can be involved in RETINOBLASTOMA and maybe in some other rare cancers
Most people have antibodies against EBV Why some populations get mononucleosis while others get tumors in not known Causes lymphoma in marmosets
Most people have antibodies against EBV Why some populations get mononucleosis while others get tumors in not known Causes lymphoma in marmosets
Latency up to 30 years
Reverse transcriptase is not a capacity possessed by normal eucaryotic cells Many features are unique to retroviruses Very unusual mode of replication which gives them the potential to transform the cell But when it comes to how these viruses cause neoplasia, they may be very similar to DNA viruses Rous sarcoma virus in chickens was the first retrovirus to be discovered. It causes an aggressive acute cancer in chickens
Even though the virus RNA is same sense (positive) as mRNA, it cannot be translated directly as it is encapsulated by proteins. Thus it must be copied via a negative sense nucleic cid (in this case via DNA). Virus contains about 10 copies of reverse transcriptase. NOTE: Both copied of single strand RNA are identical
When integrated viral genes may or may not be expressed If expressed, all are expressed at least in the simpler retroviruses (why we shall see later) Whether host polymerase makes mRNA or genomic RNA depends on the processing by the host cell splicing enzymes Nucleocapsid assembly is in the cytoplasm and reverse transcriptase is packed into the virus Virus buds through the plasma membrane where it picks up the viral glycoprotein Maturation occurs in the budded virus
To make an RNA using RNA pol II, sequences other than those to be transcribed are required. Thus the DNA “gene” is bigger than the mRNA that is transcribed from it. These extra sequences can be upstream or downstream from the transcribed portion. They include: promotors, enhancers, termination sequences. Thus these “control” sequences will be lost on reconversion of the viral genome to RNA. These sequences are necessary for transcription of mRNA but not the translation of mRNA
In fact, virus does provide its own promotors and enhancers. The clue to how it does this is in the differences in structure between the RNA and DNA forms
Enhancers may upstream or downstream. LTRs have promotors and enhancers
Note: Reverse transcriptase needs a PRIMER as does any other DNA polymerase. Prolyl tRNA is packaged in virus. This is necessary because reverse transcription starts in nucleocapsid in cytoplasm
A normal retrovirus has three genes, GAG, OPL, ENV – only these are necessary for the virus to replicate to more virus. Many, however, have another gene that allows them to transform the cell. NOTE: This extra gene is NOT necessary for a productive infection. C.f. DNA tumor viruses in which the oncogene is necessary for BOTH replication and transformation First oncogene to be discovered was the src gene of RSV. It is an extra gene at the 3’ end of the viral RNA
These viruses cannot make all of their proteins and so need a co-infecting “helper” virus Viral oncogenes have three letter names e.g. src, myb
Retroviruses that cause acute transformation (rapid onset of neoplasia) such as RSV have an extra gene. These neoplasms are usually only seen in laboratory situations. The viral oncogene causes cells to be released from their normal growth controls. These extensively characterized genes are NOT UNIQUE to retroviruses. cDNA probes against viral oncogenes show similar homologues in NORMAL eucaryotic cells . These cellular homologues are not identical to the viral oncogene and it seems that the virus has picked up a cellular gene during its evolution. The discovery of normal homologues of viral oncogenes was a great step forward in the cell biology of cancer. The cellular proto-oncogenes are a family of genes that may underlie much of carcinogenesis. BUT a cellular oncogene does not cause cancer normally. Why does it do so in a virus?
Remember: A virus has only one end: to reproduce. This means that the genome and proteins have to be made in large numbers. So many more copies of the v-onc mRNA are made than the c-onc RNA. This over expression may be the basis of the transformation that is seen
The virus is always at the same site in ALV-induced tumors. VERY SUGGESTIVE! This insertion next to a cell oncogene is similar to taking in the virus’s own oncogene. But why should c-onc be switched on as indeed it is in the tumor?
Only a few genes in the cell behave as c-oncs. They are expressed in normal cells at some time in the cell’s life. They perform a vital function. They are involved, as might be expected, in growth control. There are now about 40 of these c-oncs. A surprisingly small number that fall into several groups such as growth factors, signal transduction proteins or transcription factors
We can map genes to precise locations on chromosomes
Could the break and exchange of parts of chromosomes bring the c-onc under the control of a very active cell promotor? In Burkitt’s lymphoma there is a 8:14 translocation. Myc is at the break site on chromosome 8. What does it come next to on chromosome 14?
Thus in Burkitt’s lymphoma, the myc gene is brought under the control of a Ig promotor which is very active in this lymphocyte. Thus this is similar to putting the cellular oncogene under the control of the very active promotor in the viral LTR Proof that deregulated c-myc may be involved in tumor formation comes from transgenic mice. If a gene consisting of c-myc linked to the Ig promotor is integrated into the chromosomes of these mice there is a high frequency of lymphomas.
So far we have seen that viruses can cause cancer but it must be admitted that most cancers do not arise as a result of the mechanisms that have so far been described.
Normally, the unmutated protein binds only under special circumstances such as when a growth signal is received at the cell plasma membrane. In the heterozygote, the mutant oncogene protein (e.g. myc protein) can bind to the DNA (for example) in the absence of a signal and cause growth. Thus the mutation is dominant over the wild type as there will be mutant protein in the heterozygote that can always signal growth
Instead of being like myc which, when expressed, promotes cell division, Rb protein controls cell growth so that it is shut down. Thus when one gene is mutated so that it cannot function, there is still the other gene in the hereozygote that can produce Rb protein to control growth. Only in the homozygous mutant is there no functional Rb protein and so growth is no longer controlled.
What has this got to do with tumor viruses? The discovery of anti-oncogenes led to the elucidation of how DNA tumor viruses cause tumors.
The oncogenes of DNA viruses such as polyoma or adenovirus have been identified to early function genes and all of these seem to have characteristics in common, indeed, they have some sequence similarities and mutations in this common region abolish tumorigenicity
It is found that in adenovirus cells, the virus E1A gene makes a protein that complexes with a 105kD cellular protein. This turned out to be Rb protein. Binding Rb protein so that it cannot control growth is the same as mutating both copies of the gene for the Rb protein and so the cell continues to replicate
A similar thing happens to p53 protein in hepatitis C-infected cells and p53 is bound so that it becomes inactive. Again, lack of p53 results in loss of growth control. In papilloma-infected cells, things happen a little differently. In this case the virus makes a protease that destroys p53 protein. Thus, our knowledge of how RNA tumor viruses cause tumors led to the discovery of viral oncogenes. This led to the discovery of cellular oncogenes which in turn led to anti-oncogenes. The discovery of anti-oncogenes showed how DNA tumor viruses cause tumors.
