Oncogene

An oncogene is a kind of abnormal gene that predisposes cells to develop into cancers. Unlike normal genes, which can be turned off after being turned on, oncogenes are altered in a way that keeps them stuck in a state of constant activity. That uninterrupted action helps drive the uncontrolled growth that underlies tumors. Oncogenes can be turned on by inherited changes - ones that are passed down from parent to child - or by cancer-promoting agents that damage DNA throughout an individual's lifetime, such as UV light, hazardous chemicals and even viruses.

Cancer arises in part through damage to normal genes (known as proto-oncogenes), which can arise from exposures to cancer-promoting agents, such as UV light. That damage permanently switches the gene on, transforming it into an oncogene that helps push cells to become cancerous. The first oncogene ever identified is called src (pronounced "sarc"). Discovered in 1970, it is a component of a cancer-causing virus in chickens, known as the Rous sarcoma virus, which induces tumors in connective tissues, such as bone and muscle, in infected animals. In humans, the SRC gene was later shown to be involved in a variety of cancers, such as colon, liver, lung, breast and pancreatic cancer.

Viral oncogenes
Viral oncogenes are responsible for oncogenesis resulting from persistent virus infection. Although different human tumor viruses express different viral oncogenes and induce different
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tumors, their oncoproteins often target similar sets of cellular tumor suppressors or signal pathways to immortalize and/or transform infected cells. Expression of the viral E6 and E7 oncogenes in papillomavirus, E1A and E1B oncogenes in adenovirus, large T and small t antigen in polyomavirus, and Tax oncogene in HTLV-1 are regulated by alternative RNA splicing. However, this regulation is only partially understood. DNA tumor viruses also encode noncoding RNAs, including viral microRNAs, that disturb normal cell functions.

A BRIEF HISTORY
In 1909, a farmer brought Dr. Francis Peyton Rous, a junior faculty member then at Rockefeller University, a hen that had a breast tumor. Rous performed an autopsy, extracted tumor cells, and injected the cells into other hens, which then developed sarcoma. This was the first experimental proof of an infectious etiologic agent of cancer, and the chicken sarcoma-inducing RNA virus was subsequently named the Rous sarcoma virus. After a half-century debate on whether viruses truly cause cancer, Rous was eventually awarded the Nobel Prize in Medicine and Physiology in 1966 for his discovery of tumor-inducing viruses. It is now estimated that 20%-25% of human cancers worldwide have a known viral etiology. Early pioneering efforts on tumor-inducing viruses were mainly focused on avian and smallanimal retroviruses, a group of RNA viruses containing an RNA-dependent DNA polymerase (reverse transcriptase), and it was thought that there were no similar viruses in humans. Demonstration in 1980 of the first human retrovirus, human T-cell leukemia virus type 1 (HTLV-1), which causes adult T-cell leukemia, was therefore a landmark achievement. Later, HTLV-2, which is far less pathogenic than HTLV-1, was isolated from a hairy T-cell leukemia, but soon was demonstrated not to be the agent of the malignant hematological disease. HTLV3 and -4 have been discovered recently as new members of the HTLV family in central Africa, but their association with human diseases remains unclear.

VIRAL ONCOGENES AND THEIR FUNCTIONS E6 and E7 oncogenes in high-risk HPVs

HPV E6 and E7 from high-risk HPV types have the capacity to immortalize and transform keratinocytes and epithelial cells. Low-risk or non-oncogenic HPV E6 and E7, however, lack such biological activity. Biochemically, high-risk E6 but not low-risk E6 interacts with E6AP and the tumor suppressor protein p53 to induce ubiquitination-mediated degradation of p53. An E6 F47R mutant is defective for polyubiquitination and degradation of p53. High-risk E7 and not low-risk E7 interacts with the pRb tumor suppressor protein via the LXCXE motif in the E7 CR2 domain to promote cell cycle progression. Thus, interaction with cellular tumor suppressor proteins and perturbation of normal cell cycle control by high-risk E6 and E7 are believed to be the most important influence for malignant conversion. In this regard, E6 from HPV16 (16E6), a high-risk type, binds E6AP more strongly and drives the degradation of p53 more efficiently than 18E6 . In contrast, low-risk 11E6 has minimal binding affinity for E6AP and influences the degradation of p53 only weakly in vivo. Under hypoxic conditions, high-risk E6 also inactivates the CYLD tumor suppressor through interactions with CYLD deubiquitinase to allow unrestricted activation of NF-B.

E1A and E1B in human adenoviruses

The adenovirus genome encodes two viral oncogenes, E1A and E1B, positioned side-by-side in the left 11.2% of the genome. After an adenovirus infects a human cell, the first viral proteins that are synthesized are products of the E1A region. The full-length E1A protein (289R) is a nuclear protein consisting of 289 aa residues and has four conserved regions: CR1 at the Nterminus, CR2 and CR3 in the middle, and CR4 at the C-terminus. Large T and small t antigens in human polyomaviruses Human polyomaviruses encode two oncoproteins, large T (LT) antigen and small t (st) antigen, due to alternative RNA splicing. Both LT and st are viral nonstructural proteins expressed early in virus infection. Like SV40 T antigens, whose roles in cell transformation have been investigated extensively, human polyomaviral LT inactivates two cellular tumor suppressor
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proteins, p53 and pRB, by direct protein-protein interactions, and st also inactivates cellular protein phosphatase 2A (PP2A) through protein-protein interactions. Experimentally, coexpression of st enhances the transforming ability of Lt.

LMP1 and BARF-1 in EBV

Apparently, several restricted forms of EBV latency occur in the EBV-carrying malignancies, and modulation of LMP1 expression differs greatly depending on the latency form. In B cells with type III latency, the EBV genome expresses six nuclear antigens (EBNA-1 to -6) and three latent membrane proteins (LMP1, -2A, -2B). EBNA-2 and -5 are responsible for LMP1 expression. In type II latency, which is mainly found in Hodgkin lymphoma, T- and NKlymphoma, and NPC, the EBV genome expresses only EBNA-1 and LMPs. The expression of LMP1 is induced by IL-10 and IL-21. In Burkitt lymphoma, which has type I latency, only EBNA-1 is expressed. The oncoprotein LMP1 of EBV is a 62-kD integral membrane protein of 386 aa residues that consists of a short cytoplasmic N-terminus of 24 aa, six transmembrane domains of 186 aa, and a cytoplasmic C-terminus of 200 aa. EBV LMP1 is essential for the immortalization and transformation of human B cells, but its oncogenic activity can be downregulated by a truncated LMP1 (258 aa) . LMP1 without a ligand drives proliferation of EBV-infected B cells by signaling within the B cells similar to the signaling of the cellular CD40 receptor: both activate NF-B, AP-1, Stat-1, CD83, and CD95 by associating with molecules such as TRAFs and JAK3. LMP1's signaling, however, differs fundamentally from that of CD40, because LMP1 regulates these signaling pathways without itself being regulated by a ligand, as CD40 is. LMP1 levels vary in cells of clonal populations by more than 100-fold, which leads to multiple distinct activities of the oncoprotein. When expressed at intermediate levels, LMP1 signals through NFB to promote cell proliferation. When expressed at high levels, LMP1 inhibits general protein synthesis by inducing phosphorylation of eIF2 via activation of PERK kinase, leading to upregulation of activating transcription factor 4 (ATF4) expression, which in turn transactivates LMP1's own promoter. LMP1 activation leads to overexpression of antiapoptotic molecules, such as Bcl-2, Mcl-1, and Bcl-2-related protein A1 (Bfl-1) and blocks p53-mediated apoptosis through the induction of the A20 gene . Complementary to its proliferative function.
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Tax in HTLV
The HTLV-1 Tax protein is a 40-kD nuclear phosphoprotein that consists of 353 aa residues. To distinguish it from the HTLV-2 Tax protein (p37tax or Tax2), the HTLV-1 Tax is also called p40tax or Tax1. Tax1 and Tax2 share many characteristic properties, including in vitro immortalization of lymphocytes. HTLV-1 Tax is a transcriptional activator of the viral promoter and has been implicated in initiating transformation events leading to the development of adult T-cell leukemia, but it is not needed to maintain cell transformation, and the tumor cells from adult T-cell leukemia do not express detectable levels of Tax. This makes HTLV-1 Tax different from other cancer viruses, in which a continuous expression of viral oncogene is necessary for maintenance of transformation. However, it is clear that HTLV-1 Tax is oncogenic, because in all reported studies Tax transgenic mice were vulnerable to developing various tumors. The emerging concept is that Tax is required to initiate transformation, but viral HBZ protein and/or aberrant cellular microRNA expression appear to be needed to maintain adult T-cell leukemia when Tax is no longer expressed.

CONTROL OF VIRAL ONCOGENE EXPRESSION BY ALTERNATIVE RNA SPLICIN

The E6 and E7 genes are positioned side by side at the beginning of papillomavirus genome. The expression strategy of oncogenic E6 and E7 differs from that of non-oncogenic E6 and E7. In oncogenic HPV16 and HPV18, the two genes are expressed as a bicistronic pre-mRNA from a single promoter immediately upstream of the E6 ORF. In nononcogenic HPV6 and HPV11, the genes are expressed individually from two separate promoters. Another noticeable feature is that oncogenic E6 genes contain an intron, whereas non-oncogenic E6 genes do not. Consequently, a spliced E6 ORF is predominant in oncogenic E6 mRNAs, but does not exist in non-oncogenic E6 mRNAs, suggesting an important role of the E6 splicing in viral oncogenesis. Of note, the same promoter that drives oncogenic E6 and E7 expression is also responsible for almost all early gene expression, and the resulting early transcripts are polyadenylated by using the same early poly(A) signal. Therefore, these early primary transcripts are all bicistronic or polycistronic, and each has two or more ORFs and contains three exons and two introns.
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In HPV16, intron 1 and intron 2 of an E6E7 primary transcript each contain three alternative 3' splice sites which can be selected for RNA splicing in virus-infected cells, leading to the production of at least 14 species of mRNA transcripts with various coding potential. Intron 1 of an HPV18 E6E7 primary transcript has a single 3' splice site. Because intron 1 and intron 2 are positioned, respectively, in the E6 ORF and E1 ORF, retention of intron 1 during RNA splicing is necessary for E6 expression, whereas retention of intron 2 is needed for E1 production. E1 functions as a viral DNA helicase essential for viral DNA replication. Splicing of either intron destroys the coding of E6 or E1. Mechanistically, there are only shameful data currently available on how each intron could escape recognition by the cellular splicing machinery in order to produce these two important viral proteins. The cap-binding complex at the RNA 5' end was initially found to promotes the splicing of intron 1 of HPV16 E6E7 transcripts, but the enhanced intron 1 splicing by the cap-binding complex can be restrained by the distance of intron 1 from its RNA 5' cap. Under natural conditions, cellular epithelial growth factor (EGF) pathway regulates the intron 1 splicing of HPV16 E6E7 transcripts via Erk1/2 activation. It is possible that the oncogenic HPVs retain an intron as needed by interfering with the action of the splicing machinery using their own proteins. The findings that both HPV16 E2 and E6 act as RNAbinding proteins that suppress splicing and viral E5 regulation of EGFR expression may help us to understand this striking phenomenon.

Propagation and cultivation of viruses propagation of viruses

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printingwas used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 m/h in the 140-m lines. These stages includedinitiation
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of infection, based on G protein expression; cellcell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.

Propagation of Human Hepatitis A Virus in African Green Monkey Kidney Cell Culture:
Human hepatitis A virus (HAV) was propagated in primary African Green Monkey (Cercopithecus aethiops) kidney (AGMK) cell cultures. Three strains of HAV were used: MS-1, SD-11, and HM-175. Cells were inoculated with marmosetpassaged material or human clinical specimens and were stained by direct immunofluorescence to establish the identity of the virus. Both clinical samples and marmoset-passaged material produced immunofluorescence. HAV antigen was found scattered throughout the cytoplasm of inoculated cultures. The HM- 175 strain produced the most intense immunofluorescence. This strain of HAV had been serially passaged in cell culture seven times. Blocking experiments with paired human sera from naturally acquired HAV infections and hyperimmune chimpanzee serum from an experimentally infected animal established that the immunofluorescence was specific. The viral antigen was found to be exclusively intracellular. The interval to maximum HAV antigen expression was decreased by serial passage. The HAV strain described herein, which was recovered directly from the stool specimen of a patient with HAV in primary AGMK cell culture, may prove useful as a source of antigen for serological tests and as a candidate vaccine strain. Hepatitis A virus (HAV) was only recently propagated in cell culture for the first time (13). HAV most closely resembles the picornaviruses, especially the subgroup of enteroviruses (3, 4, 7, 14, 16, 17; Y. Moritsugu, T. J. W-K Shih, T. Kakefuda, S. M. Feinstone, J. L. Gerin, and R. H. Purcell, submitted for publication). It may share properties with many of these viruses, such as the coxsackievirus A group, some of which are also difficult to propagate in vitro. Since HAV does not seem to be produced in large quantities from tissue culture cells (see below) nor to produce recognizable cytopathic effect, the development of in vitro culture systems could not have occurred until after the development of an adequate detection system, in this case,
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immunofluorescence (IF). By utilizing the recently developed HAV-specific IF system (12), Provost et al. (13) demonstrated that HAV passaged 31 times in marmosets could replicate in cell culture. Since it was not clear if the important step in the in vitro cultivation of HAV was the adaptation to marmosets, the cell culture substrate used, or the IF detection system, we have attempted the in vitro cultivation of HAV obtained directly from human clinical specimens as well as after serial passage in marmosets.

Cultivation of viruses
As the viruses do not reproduce independent of living host cells, they cannot be cultured in the same as bacteria and eukaryotic microorganisms. However, the cultivation of viruses can be discussed under following heads: (i) cultivation of animal viruses, (ii) cultivation of plant viruses, and (iii) cultivation of bacteriophages.

Cultivation of Animal Viruses (i) In Animal Cells

Suitable living mammals (such as sheep or calves or rabbits) are selected for cultivation of viruses. The selected animals should be healthy and free from any communicable diseases. The specific virus is introduced into the healthy animals. The site of administration varies according to the type of virus is allowed to grow in the living animal. At the end of incubation period, the animals are slaughtered and washed thoroughly and viruses are obtained from them.

(ii) In Chick-Embryo
The animal viruses can be successfully cultivated using chick-embryo technique. In this method fertile hen eggs are selected. Eggs must not be more then 12 days old. To prepare the egg for virus cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill. After inoculation, the drill hole is sealed with gelatin and the egg is then incubated. Viruses may be able to region. For convenience, the myxoma virus grows well on the chorioallantoic membrane, whereas the mumps virus prefers the allantoic cavity. The infection may produce a local tissue lesion known as pock, whose appearance often is characteristic of the virus.
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(iii) In Vitro Culture (Tissue Culture Technique)

More recently developed in vitro cultivation of animal viruses has eliminated the need to kill the animals. This technique has become possible by the development of growth media for animal cells and by the availability of antibiotics which prevent bacterial and fungal contaminations in cultures. Cultivating animal viruses using tissue culture technique involves following three main steps: Cultured cells Primary Heterogeneous many cell types Closest to animal Technical hassle Diploid cell strain Relatively homogeneous fewer cell types Further from animal Technically less hassle
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 Continuous cell line Immortal Most homogeneous Genetically weird furthest from animal Hassle free Suspension or monolayer

Monolayer Preparation. Live tissues of vital organs (e.g., heart or kidney) are taken and the cells are separated from the tissue by digesting the intracellular cement substance with dispersing agents such as trypsins or collagenase or ethylenediaminetetraacetic acid (EDTA). The cell suspension is passed through screen filters so that the coarse particles are removed from the separated cells. The cells are washed free of dispersing agents. The cells are centrifuged if required and resuspended in nutrient medium contained in glass or plastic vessels. The composition of medium and other conditions of incubation depends on the type of cells used. Upon incubation the cells quickly settle and attach firmly to the bottom of the flask. If undisturbed, these cells grow and spread to form monolayers.

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Clonal Cell Line Preparation

The monolayer cells are first removed and washed with saline solution devoid of calcium and magnesium ions and then added to the dilute solution of EDTA (1: 3000) to chelate intracellular magnesium or calcium ions. After sometime, the loosened cells are shaken and resuspended in growth medium in fresh culture vessels and incubated. The cells are cultivated under 5% CO2 condition. The cultures of cell obtained so are called diploid cell strain. It is extremely difficult to distinguish primary cell and the diploid cell strain. On repeated subculturing, each cell starts multiplying to form separate colony. If each colony is removed and cultivated separately, it forms pure culture. These bunches of cells from single cell is called clonal cell lines.

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Infection with Virus The clonal cell lines suspended in suitable media are infected with any desired virus which replicates inside the multiplying cells. If the virus is virulent, they cause lysis of cells and virus particles are released in the surrounding medium. These newly produced virus particles (virions) infect the adjacent cells. As a result localized areas of cellular destruction and lysis (called plaques) often are formed. Plaques may be detected if stained with dyes, such as neutral red or trypan blue that can distinguish living from dead cells. Viral growth does not always result in the lysis of cells to form a plaque. Animal viruses, in particular, can cause microscopic or macroscopic degenerative changes or abnormalities in host cells and in tissues called cytopathic effects, cytopathic effects may be lethal, but plaque formation from cell lysis does not always occur.

Cultivation of Plant Viruses

There are several methods of cultivation of viruses such as plant tissue cultures, cultures of separated cells, or cultures of protoplasts, etc. Viruses also can be grown in whole plants. Leaves are mechanically inoculated by rubbing with a mixture of viruses and an abrasive such as carborundum. When the cell wall is broken by the abrasive, the viruses directly contact the plasma membrane and infect the exposed host cells. (The role of the abrasive is frequently filled by insects that suck of crush plant leaves and thus transmit viruses.) A localized necrotic lesion often develops due to the rapid death of cells in the infected area. Even when lesions do not arise, the infected plant may show symptoms such as change in pigmentation or leaf shape. Some plant viruses can be transmitted only if a diseased part is grafted onto a healthy plant.

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