| MicrobiologyBytes: Virology: Transformation | Updated: October 19, 2004 | Search |
The genes responsible for transformation in these viruses have no cellular homologues. They are true viral genes required for viral growth. Most cells in the body are quiescent and not synthesizing DNA for replication. DNA viruses need to make DNA in order to replicate and many of them are partially dependent on the cell to do this. They therefore modify gene expression in the host cell to favour DNA synthesis and cell replication e.g adenovirus in terminally differentiated epithelial cells, these cells must be pushed into S phase for viral replication. The normal result of an infection is a lytic cycle resulting in the death of the cell.The aberrant result of a non-permissive infection is rarely (10-5) transformation. This inefficiency reflects the lack of a specific mechanism for genome integration. The viral proteins responsible for transformation generally have a nuclear location. They have proved to be an interesting tool with which to study cell growth control mechanisms. |
Have small genomes ~5-10kb and have been extensively studied. The family includes SV40, and polyoma virus. These viruses can interact with cells in one of 2 ways:
SV40 was originally identified as a non-cytopathic virus in rhesus monkeys which could produce a lytic infection in green monkey cells and cause tumours in hamsters. The possibility that transformation was a result of integration was ruled out early on and attention focused on viral (onco)genes.
SV40 codes for two "T-" or tumour antigens 94K and 17K. They are the result of differential splicing of a single transcript. Small T seems to be dispensable for virus growth in culture and for transformation. Large T acts as a switch from early to late transcription for the virus.
Studies show that large T is multifunctional in nature, both with respect to its viral functions and its transforming functions.
Properties of SV40 Large T antigen:
Molecular genetic analysis suggests that N-terminal 140 or so amino acids of large T is involved in transformation. Specifically a region between 105-114. Sequence homology with E1A transformation domain from adenovirus. Binds to Rb. Mutants in this region are defective in induction of focus formation. p53 binding site is located at the C-terminus. Nuclear transport defective mutants of large T have been analysed, when such a mutation is transfected into primary cells it does not transform suggesting a nuclear function. However, transformation of NIH 3T3 cells is as efficient as the wild type gene. Here it is assumed to be functioning in the cytoplasm. i.e. consistent with a multistep model of transformation.
Mechanism of action is probably via an interaction with tumour suppressor genes. Rb-p105 is a phosphorylated nuclear protein which modulates transcription. Rb gene identified in individuals who develop retinoblastoma early in life. p53 also modulates transcription. T antigen association with these 2 proteins is postulated to overcome the inhibition of cell growth.
Fig 5.16 These are large d/s DNA viruses with genomes in excess of 100kb. Human serotypes transform rat cells and some cause tumours in hamsters but natural infections have not been shown to cause transformation. The viral genome integrates into cellular DNA. The 3' 2800bp of the genome is involved in transformation, and is known as E1 region (3' end r strand). Two transcripts are associated with this region, E1A and E1B. Both give rise to multiple RNAs and proteins:
The E1A protein is nuclear and phosphorylated at multiple serine residues, acidic and proline rich.
3 functions have been assigned to specific domains in the proteins:
Transactivation is not due to direct DNA binding but association with other transcription factors such as AP1. As with the T antigen of SV40, there is great interest in its interaction with Rb protein and other cellular proteins.
The E1b protein can interact with p53.
Genuine human tumour viruses which may be responsible for 10% of human cancers.
Diagnosis and treatment: A wart is seldom misdiagnosed, most regress spontaneously. Importance depends on location, skin wart may be unsightly but a wart on the vocal chord may be life threatening. Traditional therapy is application of caustic agent, cryotherapy, 5 flurouracil and surgery. Interferon therapy useful for genital warts of less use for oral warts. Prevention, spread by infectious virions contacting minor epithelial abrasions. No vaccine imminent, infection not thought to invoke a strong and lasting immune response. Genital infection best avoided by safe sex practices: it is estimated that about 80% of the adult population are infected many subclinically. The fact that only a small number of "high risk" infections leads to cancer suggests that HPV infection alone is not sufficient to cause cancer but is one significant step towards it (90% of all cervical cancers contain HPV DNA). Infants which passage through an infected birth canal may manifest juvenile onset laryngeal papilloma most before the age of 5, life threatening. Tends to recur and patients require surgery frequently, some every 2 weeks. Immunosuppressed transplant patients also prone to clinically troublesome warts.
Papillomaviruses have 8 kb genomes, are epitheliotropic and cause benign proliferations and also cancer. More than 70 pathogenic human strains have so far been identified. 2 main groups, cutaneous and mucosal, a substantial number infect the anogenital tract and may cause penile, vulval, and anal warts and penile vulval and cervical cancer. Transmission of these papilloma viruses is sexual and the diseases are sexually transmitted. These infections are very rare in virgins. Infection is directly correlated with the number of partners a couple has had unprotected sex with. The oral mucosa similarly sensitive to these genital viruses. Several viruses are exclusively infect the oral mucosa. 90% of all cervical cancers contain HPV DNA.
The E6 and E7 genes of HPV 16/18 are expressed as proteins in human tumours and tumour derived cell lines. E6/7 DNA can transform NIH3T3 cells. E7 is a cytoplasmic serine phosphoprotein, and has been shown to be a transcriptional transactivator and transforming protein. E7 binds Rb protein-presumably moves to nucleus. E6 binds to a cellular protein called E6-Ap. This complex then binds to p53. E6-Ap is a ubiquitin ligase. Cellular enzymes load it with activated ubiquitin molecules which are transferred to p53. Ubiquitin loading of p53 targets it for degradation by proteasome mediated proteolysis. The forced entry into S-phase in conjunction with genomic instability, resulting from p53 degradation may lead to malignancy. E6/7 proteins from 'low risk' HPV strains appear not to associate with Rb and p53. Expression of E6/7 antisense constructs reduces cell growth indicating that E6/7 may not only participate in initiation but also maintain the proliferative and malignant phenotype.
E7 can also interact with transcription factors such as jun and fos, upregulating Ap1 responsive promotors
Other observations suggest that E6-7 gene expression is necessary but not sufficient for malignant growth:
Most herpesviruses are not proven to be responsible for any human cancers, although they may be a cofactor with papilloma for cervical cancer. However, HHV8 has recently been implicated as the causal factor for Kaposi's sarcoma, KS. The virus is present in tumour cells, tracks tightly to and precedes the presence of disease. Latent infection but a switch to the lytic phase may be responsible for the formation of lesions. KS is a disfiguring skin cancer which is particularly common in AIDS patients. It is a peculiar neoplasm containing many different cell types. Possibly paracrine in origin. Spindle cells are dominant but there are also infiltrating inflammatory cells and permeable neovascular elements. Extravasion of erythrocytes gives rise to the characteristic purple appearance of the lesions In the immunocompetent the tumour is indolent and localised. In the immunocompromised it is more widespread, disfiguring and possibly fatal. Restoration of the immune system can result in arrest and even remission. The spindle cells are not fully transformed but do grow and secrete proinflammatory and angiogenic factors. Cause vascularised tumours in nude mice. HHV8 appears to be the agent responsible for promoting spindle cell growth. This virus is a member of the lymphotropic subfamily of the herpes virus, as is EBV. As well as being present in KS, the viral genome can also be found in:
Epstein Barr Virus (EBV) 90% of adults in W.Europe and N. America carry EBV. Infection persists for life with virus regularly shed by salivary gland. Salivary fluid transmits the virus e.g. infected eating utensils and kissing. It is a mark of how well adapted to us this virus is,that it can persist for a lifetime in an asymptomatic state.T cells effectively control it.In vitro it is the most potent transforming virus known.Definition of transformation in this system is the conversion of non-dividing B lymphocytes from (EBV-) individuals to indefinitely proliferating lymphoblasts. It is linked to at least 4 different types of tumour:
A link with Burkitt's lymphoma was first established when virus isolated from cultured Burkitt's cells, and linked to the disease sero-epidemiologically. EBV DNA can be isolated from tumours, viral specific antigens can be detected in the tumours, infection with EBV induces a lymphoma like disease in new world primates. In vitro EBV immortalization of B cells further supports the link.
The mechanism by which the virus transforms cells is still uncertain. The transforming gene(s) have not been unambiguously identified (large virus). Possible candidates include EBNA-1 (Epstein Barr nuclear antigen) and EBNA-2. EBNA-1 associates with a transcriptional enhancer element in the EBV origin of replication. Also suggested that EBNA-1 activates the lymphoid recombinase RAG genes. Activation of these genes may be required for viral integration into the host genome. It could also result in chromosomal rearrangements and translocations. EBNA-1 Tg mice have an increased incidence of lymphoma. EBNA-2 is a transcriptional transactivator for both viral and cellular genes. It upregulates the LMP-1 and LMP-2 genes, as well as markers for B cell activation e.g CD23 as well as CD21, the EBV receptor. Another candidate is LMP, the latent membrane protein which acts as a constitutively activated receptor. It is the only latent viral gene product which can transform cell lines in vitro. It induces the expression of many cellular genes including markers for B cell activation. LMP-1 interacts with cellular proteins that transduce signals from the TNF family of receptors e.g TRAFF and TRADD (TNF receptor associated factor and TNF receptor associated death domain). It prevents p53 induced apoptosis and bypasses Rb function. Seems to act as a constitutively activated receptor via NFKB. So this oncogene is not directly interfering with tumour supressor proteins and nor is it directly related to a cellular oncogene; it seems to mimic the structure of an activated component instead.
The contribution of EBV in nasopharangeal cancers, Hodgkin's lymphoma and B lymphomas in immune suppressed individuals remains to be elucidated, other cofactors must be involved.
There is no effective chemotherapy for EBV infections. BL multifocal at time of diagnosis so chemo is therapy of choice. Remission induced in 80% of patients treated.NPC is a malignancy of the squamous epithelium in the posterior nasopharynx. NPC is treated by surgery and radiotherapy. Survival rates good if it has not reached the supraclavicular fossal lymph nodes. If it has then less than 20% survive 5 years. Good example of the interplay of viral, genetic and environmental factors. Consumption of salted fish in endemic regions is high. These are thought to contain high levels of phorbol esters and nitrosamines. Genetic studies have shown a loss of heterozygosity in several chromosomal regions thought to contain a susceptibility locus. Most carcinomas have a prolonged dysplasia-carcinoma in situ phase. But not in NPC, viral gene expression is thought to result in a rapid clonal expansion of cells and invasive neoplasia.In post transplant lymphoma a reduction in immunosupression therapy may induce regression.
Mutations are not found in Rb or p53 in EBV associated tumours. Presumably viral gene expression interfers with the function of these proteins in another way
More than 200 million infected worldwide. 2 million die annually of which 700 000 are due to development of a monoclonal hepatocellular carcinoma (HCC). HBV is sexually transmitted but much more infectious than HIV. 30% of the infected individuals develop a mild acute infection within a few months. Symptoms are managed during the acute phase. It is not known what can be done to prevent the development of a chronic infection which may be asymptomatic or incapacitating.
HCC develops in 2-10% of infected patients after several decades of chronic liver disease and cirrhosis. Accounts for 30% of viral induced cancers and 5% of all cancers. The risk of ceveloping HCC is increased 100 fold following chronic infection with this virus. There is a seroepidemiological correlation between HCC and HBV infection throughout the world, except curiously for Greece. Viral transcripts can also be identified in transformed cells.
The HBV genome persists in the great majority of tumour cells. ORF X which codes for a transactivator protein is maintained. Transfection of ORF-X into NIH3T3 cells induces tumours in nude mice. Liver cancer develops in mice transgenic for ORF-X. In humans a pleiotropic transactivation of cellular genes by ORF-X and Pre S2 is thought to activate PKC/Raf signalling pathways. This activates the AP1 and NFKB transactivator functions which in turn control genes relevant for proliferation. Also find modifications in the short arm of C11 and the long arm of C13 in HCC. Perhaps tumour suppressing genes are located at these positions ? Point mutations in p53 have been reported in HCC. Continuous liver cell regeneration may allow the accumulation of mutational events that lead to the emergence of an HCC clone.
A range of hepadnaviruses also infect birds e.g. woodchuck hepatitis virus-induces cancer a year or so after exposure. In these animals no cirrhosis develops and there is little hepatitis. But the viral DNA is rearranged and the tumour is monoclonal. This suggests that specific viral functions are more important for initiation and maintenance of proliferative changes than regenerative events. Cis activation of c-myc and N-myc is almost always observed.
Control: Medical workers are susceptible. Stable virus, mucosal and epidermal routes of entry. Infection can be prevented by avoiding direct contact with body fluids. A vaccine is available, not routinely administered in the UK! But this virus will remain a serious public health problem for years to come, despite the vaccine. Therapy is liver resection, transplantation, or ethanol injection into the tumour.
© MicrobiologyBytes 2004.