1.1 Background
About 12% of human cancers globally are caused by oncoviral infection with more than 80% of them found in the developing countries. Human viral-associated cancers are of public health importance and suitable for immunoprophylaxis, and targeted therapies. However, understanding and managing viral-associated cancers still faces formidable challenges as result of limitation in animal models of the disease, disparate nature of these cancers, and the different type of viruses that caused them as well as the complex nature of the virus-host interaction which leads to the development of cancer. Oncoviruses can be either DNA or RNA viruses (Table 1). DNA tumor viruses have a DNA genome that is transcribed into RNA which is then translated into proteins. They have two life styles: 1. in permissive cells, all parts of the viral genome are expressed. This leads to virus replication, cell lysis and cell death, and 2. In cells that are non-permissive for replication viral DNA is usually but not always integrated into the cell chromosome at random sites. Only part of the viral genome is expressed. This is the early, control functions of the virus. Viral structural proteins are not manufactured and no progeny virus is released. The RNA tumor on the other hand differs from DNA tumor because their genome is RNA but they are similar to DNA tumor viruses because the genome is integrated into the host genome. Since RNA makes up the genome of the mature particles, it must be copied to DNA before it is integrated into the host cell chromosome. This strategy is against the central dogma of molecular biology in which the DNA is copied into RNA. DNA tumor viruses has been implicated in the etiology of human cancers including human papillomavirus (HPVs), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), hepatitis B virus (HBV), and Merkel cell polyomavirus (MCV). Among the RNA viruses, hepatitis C virus (HCV) and human T cell leukemia virus type-1 (HTLV-1) are associated with human cancers. Infection with human immunodeficiency virus (HIV) is associated with cancer incidence although immunodeficiency maybe a contributing factor. According to the International Agency for Research on Cancer (IARC), infection with HPV 16,18,31,33,35,45,51,52,56,58,89 and 66 are the lead cause for cervical cancer. EBV was first identified in 1964 and was the first recognized herpesvirus to be identified as oncogenic in human. EBV is associated with 4 types of malignancies: Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma and non-Hodgkin’s lymphoma linked with post-transplant or immunosuppression by HIV.KSHV is also referred to as human herpesvirus -4 (HHV-4). KSHV is also known as HHV-8. In the early 1990s, it was reported that Kaposi’s sarcoma (KS) was one of the apparent clinical manifestations of acquired immunodeficiency syndrome (AIDS). KSHV was subsequently reported from a case of AIDS- associated KS in 1994. HBV is single stranded RNA virus that is transmitted mostly via contaminated blood or unsafe medical procedure. The World Health Organization (WHO) estimates that about 170m people are infected worldwide although the number keeps increasing at an alarming rate. It is one of the causes of liver cancer (hepatocellular carcinoma, HCC), the sixth most common cause of cancer in the world and the third in terms of mortality. It is estimated that 54% and 31% of the global cancer burden are attributed to HBV and HCV infections respectively. The development of HCC is generally slow, over a period of more than 30 years after infection with HBV or HCV. HTLV-1 was the first reported retrovirus isolated in 1980 and 1981 from American and Japanese patients suffering from adult T-cell leukemia (ATL). The causal relationship between the virus and malignancy has been established. It is transmitted mostly via mother to child through breastfeeding; sexually via partners or iatrogenically through transfusion of blood products. It is estimated that about 15-20 million people are chronic HTLV-1 carriers of which up to 5% are at risk of developing ATL. A human T cell leukemia type II (HTLV-II) has been described. Unlike HTLV-I, it shows tropism for CD4+ lymphocytes, preferentially infecting CD8+ lymphocytes. HTLV-II is endemic in a number of geographic regions such as North Africa, parts of North and South America. There is however limited evidence to link it to human diseases despite it prevalence. Most cases of HTLV-II infection are associated with hematologic disturbances such as pancytopenia, atypical hairy cell leukemia, lymphatic leukemia, large cell lymphoma, and mycosis fungoides. HTLV-II infection may also produce neurological disorders such as cognitive dysfunction. With increasing incidence and prevalence of viral associated cancers, it is important to identify more research theme on HTLV-II.
Historically, Francis Peyton Rous on 1 October 1909 started search for oncoviruses when he initiated a cancer virus transmission study at Rockefeller University, USA on a 15-month old hen which had sarcomatous chest tumor that Rous successfully transplanted into other chickens. By 1911, he had successfully showed the cancer could be transmitted via cell- free tumor extracts. He concluded that it might probably be a virus that caused that tumor. Earlier in 1908, 2 Danish scientists Oluf Bang and Vilhelm Ellerman had published a paper on viral transmission of avian erythoblastosis. Rous gave up his studies on viral cancers until 1930s when mammalian tumor cancer was described. Rous returned to viral tumor biology using cottontail rabbit papillomavirus in 1934 with his colleague Richard Shope. In the 1950s, interest in viruses as cause of cancer grew after Ludwick Gross had discovered an acute transforming murine retrovirus. During the same period, mouse leukemia virus and polyomavirus were discovered. Rous was awarded the Nobel Prize in 1966 to Rous for discovering Rous sarcoma virus. In humans, EBV was the first tumor virus to be discovered in the 1960s and 1970s. In 1970s, epidemiological studies linked HBV infection to HCC. HPV was associated with cervical carcinoma in a proposal by zur Hausen in the 1970s. This led to the development of Cervarix and Gardasil, two anticancer agents that protects against infection with HPV16 and HPV18, the cause of most cervical cancer. In the same 1970s, clustering cases of leukemia in Southwest Japan led to the isolation and description of retrovirus which was shown to be identical to adult T-cell leukemia. In 1989, John Michael Bishop and Harold E. Varmus were awarded the Nobel Prize for discovering the viral oncogene c-Src. Advances in molecular techniques had positive impact in the field of oncovirology. Two new oncoviruses were discovered. In 1994, Chang et al using a PCR-based technique discovered Kaposi’s sarcoma-associated herpesvirus (KSHV). Later Merkel cell polyomavirus (MCV) was discovered. It is the only proven human polyomavirus with proven oncogenic ability among the many human polyomavirus. MCV was discovered among patients with Merkel cell carcinoma using digital transcriptome substraction (DTS). In 2008, the Nobel Prize was awarded to zur Hausen for discovering that high-risk HPV causes cervical cancer and discovery of HIV, a virus that does not directly initiate cancer but initiated the stage for cancer growth through immunosuppresion by Luc Montagnier and Francois Barre-Sinoussi.
|
Virus |
Associated cancers |
Mode of transformation |
Mechanism of carcinogenesis |
|
HPV |
Cancers of the cervix, anus, penis, vulva, vagina, oropharynx |
Direct |
Production of the proteins E6 and E7 |
|
HBV |
HCC |
Indirect |
Chronic inflammation |
|
EBV |
Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, non-Hodgkin lymphoma associated with post transplant or HIV immunosuppresion |
Direct |
Production of viral oncoprotein during lytic infection |
|
HCV |
HCC |
Indirect |
Chronic inflammation |
|
KSHV |
Kaposi’s sarcoma, PEL, MCD |
Direct |
Production of viral oncoprotein during lytic infection |
|
MCV |
MCC |
Direct |
Viral genes large and small tumor antigen |
|
HTLV-1 |
ATL |
Direct |
Production of viral oncoprotein Tax |
|
HIV |
Kaposi’s sarcoma |
Direct |
immunosuppresion |
Table 1: Characteristics of some oncoviruses
1.2 Prevalence of some Oncoviral Infections
EBV is highly prevent around the globe with an estimated 90% adults infected with the virus. It is estimated that around 5.5 billion are infected with EBV globally. Two types of major of EBV have been identified with different in geographical distribution. EBV-2 is more common in Africa and homosexual men. HBV infects more than 2.0 billion worldwide and more than 300 million are chronic HBV carriers with an estimated 1 million deaths annually from HBV- associated liver diseases including severe complications such as liver cirrhosis and HCC. The prevalence is highest in Sub Saharan Africa, the Amazon Basin, China, Taiwan, and several countries in Southeast Asia. In areas of high endemicity, the life time risk of HBV infection is more than 60% with more infections acquired via perinatal and child-to-child transmission where the risk of becoming chronic infections is greatest. Vertical transmission is mostly predominant in China, Korea and Taiwan while in Sub Saharan Africa, child-to-child transmission is most common.
On the other hand, HCV infects around 150 million people worldwide with an estimated 2.2 % prevalence. The estimate of HCV ranges from < 0.1% in UK to 15-20% in Egypt. The high prevalence of HCV infection is found in Mongolia, northern Africa, China, Pakistan, Southern Italy, and some areas of Japan. About six major genotypes of HCV have been described. These include: genotype 1, genotype 2, genotype 3, genotype 4, genotype 5 and genotype 6. About 75% of Americans with the virus have genotype 1 (subtype 1a or 1b), and 20-25% have genotype 2 or 3, with small numbers infected with genotypes 4, 5, or 6. Genotype 4 is much common in Africa while genotype 6 is common in Southeast Asia. HCV has two major route of transmission: injecting drug use and iatrogenic exposure through transfusion, transplantation, and unsafe therapeutic interventions.
HIV infection is found in an estimated 36.9 million people globally at the end of 2014. An estimated 0.8% of adults aged 15-49 years are living with the virus worldwide. Sub Sahara Africa continues to house the biggest burden with 70% in 2014. After Sub Sahara Africa, the Caribbean, East Europe and Central Asia are the most severely affected. There were 2.0 million, among them 0.39 million children who were newly infected with HIV at the end of 2014. HIV-1 is transmitted via three major routes: sexual intercourse, blood contact and mother-to-child transmission.
Cervical cancer is the most common cervical cancer among women in the developing countries with more than 85% of the global cervical cancer-associated deaths occurring in these countries. Molecular epidemiological analysis has shown that HPV infection is the major cause of cervical cancer. More than 200 HPV genotypes have been described and characterized based on nucleotide sequence relating to the L1 gene which codes for the major HPV capsid protein. Based on their oncogenic potential via association with cervical cancer and precancerous lesions, HPV have been grouped into two groups: high –risk (HR) genotype that causes cervical neoplasia, and low risk (LR) genotype that causes mild dysplasia. The prevalence of HPV infections in women within the general populace varies considerably within countries and regions, and within regions, ranging from 1.6 – 41.9%; for e.g. a study found that the overall HPV prevalence among Arab women in Qatar was 6.1%.
The human T-cell lymphotropic virus-1 (HTLV-1) is the only retrovirus known to directly cause cancer. HTLV-1 is endemic in South Japan, Central Africa, North Eastern South America, the Caribbean, and South Eastern United States. It is also found among IV drug users in the US and Europe and foci in Middle East and Melanesia. The prevalence of HTLV-1 increases gradually with age, especially among women in all the highly endemic areas. It is transmitted by mother to child, sexual transmission and transmission through contaminated blood products. Although lot of data is lacking in large areas, it is estimated that about 5-10 million of people are infected with the virus. HTLV-1 is the major cause of ATL, HTLV-1’ associated myelopathy / tropical spastic paraparesis (HAM/TSP), and uveitis. Due to lack of data, it is important to study on HTLV-1 and diseases outcome such as urinary tract disorders, increased susceptibility to infection, etc. HTLV-1 has subtypes including subtype A which include the prototype sequence from Japan and found mostly in endemic areas worldwide; subtype B, D, and F found in Central Africa; and subtype C found in the Melanesia
KSHV is the cause of KS. The prevalence of KSHV infection varies, from about 1-3% of blood donors in North America to more than 70% in Africa where it is endemic. The prevalence of KSHV infection approximately mirrors the prevalence of KS. A relatively high seroprevalence of KSHV has been described among injection drug users and women with multiple sex partners, although the incidence of KS among these groups is negligible. KSHV seroprevalence has also been reported to be high among family members of KSHV-seropostive persons. In areas where the virus is endemic, the highest degree of concordant seropositivity is found between mother and child or pair of sibling, and seropostive. Based on these data, the suggestion is vertical or parenteral transmission of KSHV is rare and inefficient but the high prevalence of the virus among children in most endemic regions also argues against the sexual contact as the predominant mode of transmission.
MCV is the cause of approximately 80% of MCC, a rare and highly aggressive skin cancer. It was discovered in 2008 by researchers at the University of Pittsburg. It also referred to as cutaneous apudoma, or primary neuroendocrine, and trabecular carcinoma of the skin. MCC is common among the older white men and largely absent in populations less than 40 years. The estimated annual incidence of MCC in the US is about 470 cases per year but of late there has rise in cases of MCC especially in organ transplant patients, reduced immunity, and lymphoma. In people with HIV, the relative risk for the tumor is 13.4 when compared to the general population. The tumor is most often located in the sun-exposed skin of the head, neck and the extremities.
1.3 Does Viruses cause cancer? Issue of causality
In medical investigations, the classic standard for measuring causality is based on Koch’s postulates. In summary, the postulate states that for a pathogen to be regarded as the cause of a disease, then: 1. The pathogen should be found in all cases of the disease but not in healthy individuals unless there can be asymptomatic carriers, 2. The pathogen must be isolated from the disease and propagated in culture, 3. Pathogen from the culture should cause the same disease when re-inoculated, and 4. The pathogen must be re-isolated from the inoculated host with the disease and be identical to the original agent. These postulates are difficult to apply in the cases of human viruses and cancer. First, it is known that in most cases there is a long period of latency between primary viral infection and the manifestation of cancer. In HTLV-1 infection for example, the latency period between infection and the onset of acute T-cell leukemia is to the order of decade, and in addition only a fraction of those infected will go on and develop ATL. Also some viruses establish subclinical infection so it will be difficult to ascertain the time of infection. Another issue is oncoviral infection is widespread but associating it with cancer is rare. For example, seroepidemiological studies shows that 63% to 75% of the population in the US is infected with MCV but the incidence of MCC is 0.17/100,000 to 0.34/100,000. The outcome of viral infection depends on host factors such as the immune status, for example the immunosuppressive ability of HIV-1 is a major predisposing factor to KSHV. In addition, other viruses require cofactors to initiate the cancer process; for example in HPV, the cofactors required for cervical cancer include hormonal contraceptive, coinfection with other agents such as Chlamydia, smoking, nutrition, etc. Some oncoviruses such as HSV irreversible integrate into the host genome during pathogenesis thereby making it difficult to be cultured infectious progeny. Most viruses lack animal model and some such as MCV even lacks cell culture system. Furthermore, other cancer-associated viruses utilize different mechanisms during the process of carcinogenesis; for example, in HPV-associated cervical cancer, the virus promotes chromosomal instability thereby contributing to cellular genetic changes. The role of some of the viruses in cancer pathogenesis would be discussed in the various chapters.
So how do we solve this problem? A number of approaches have been suggested based on criteria that define environmental causes, consistency, specificity, temporality, plausibility, etc. In summary, the guidelines proposed for a given virus to be regarded as the cause of a human cancer are:
1. The geographical distribution of viral infection should match that of cancer after adjustment for other cofactors.
2. Viral markers such as antiviral antibody should be higher in cases of cancer than in controls.
3. Viral markers being present should precede the tumor and have an incidence that matches the incidence of the tumor.
4. Prevention of viral infection though intervention such as vaccination should result in decrease in incidence of the tumor.
5. The virus should exhibit transforming properties with human cells in culture, and
6. The virus should induce tumors in animals and this should be preventable when viral neutralization techniques are applied.
In general these can be complex and depends on the virus. However in order to accept these criteria, more research on the virology, epidemiology and molecular biology of these viruses is needed.
1.4 Viral Carcinogenesis
Molecular biology has played a significant role in the discovery of many mechanisms utilized by oncoviruses to initiate the carcinogenesis processes by altering the function of cellular targets which significantly play important role in the development of cancer. Carcinogenesis is a multistep process involving pre-initiation, initiation, promotion and metastasis. With oncoviruses, three mechanisms have been implicated in the carcinogenesis process. These are direct, indirect as a result of chronic inflammation, and indirect through immunosuppresion. The direct carcinogens include EBV, HPV, HTLV-1, and KSHV; the indirect carcinogens through chronic inflammation include HBV and HCV; the indirect carcinogens through immunosuppresion include HIV-1. Direct viral carcinogens possess the following characteristics: 1. the entire or partial genome of the virus is usually detected in each cancer cells. 2. The virus expresses a number of oncogene that interacts with cellular proteins to disrupt the checkpoints of cell cycle, inhibits apoptosis, and DNA damage response, cause genomic instability, and induce cell immortalization, transformation, and migration. For e.g. HCV cause HCC through chronic inflammation which leads to the production of chemokines, cytokines, and prostaglandins that is secreted by the infected cells and/ or inflammatory cells. Chronic inflammation also results in the production of reactive oxidative species with direct mutagenic effect to deregulate the immune system thereby promoting angiogenesis, an essential factor for neovascularization and survival of tumors. HIV-1 infected individuals are at risk of developing cancers caused by another infectious agent through immunosuppresion leading to increased replication of oncoviruses such as EBV. Although antiretroviral agents have reduced the risk to these cancers, the risks still remain high around the globe. Only a proportion of those infected with oncoviruses develop cancer. This means there are cofactors which play some roles in the carcinogenesis process of oncoviruses. As stated earlier, carcinogenesis is a multistep process; which means there might be multiple risk factors; for e.g. viral factors, host factors, and environmental factors. The host and viral factors will be discussed later but the environmental factors include nutrients, immunosuppresion drugs, co infection with other pathogens, etc. Other factors might be involved but are yet to be elucidated. Alteration of certain genes (through mutation) involved in cellular functions leads to malignant transformation; the best documented is the tumor suppressor protein p53 which will be dealt with in details later. Several viral oncogenic proteins and factors have been described. This section will highlight on a few of them. Other proteins associated with such processes will be discussed later.
1.4.1 Hypoxia-inducible factor 1 (HIF-1): HIF proteins are major component of the innate hypoxic stress response in non-cancerous cells, acting as multitude of genes needed for adaptation under low oxygen. So far three HIF isoforms has been described, viz. HIF-1, HIF-2, and HIF-3. Available data shows that activation of HIV-1 transcription factor is a pathway mostly affected by human oncoviruses. Bersten et al described the component of HIF-1. Briefly, it is a heterodimer consisting of α and β subunit. The dimer is a member of helix loop helix-PER-ARNT-SIM (bHCH-PAS), a family of transcription factors associated with the development of cancer. In normal, non-hypoxic cells, HIF-1α is synthesized continually and degraded while HIF-1β is also continuously expressed to levels that remain constant with the nucleus. HIF-1 activity is therefore dependent on the regulation of HIF-1α. HIF-1α mRNA is expressed and its levels are similar for most cells studied between hypoxic and normoxic condition. However some cell types such as HCC Hep3B cells however, there is increase in HIF-1α transcription under hypoxic condition. Other in vivo studies showed that there is the potential of environmental hypoxia inducing HIF-1α transcription. Therefore most studies supports the suggestion that oncoviruses enhances HIF-1α levels through the modulation of its transcription, translation, or stabilization. However evidence is lacking on whether HIF-1α target gene is necessary for malignant transformation. Also, a complete host-virus interaction, effects of viral genomic variation, and the potential therapeutic benefits of utilizing HIF-1α in viral carcinogenesis require more investigations.
1.4.2 Proto-oncogene: An oncogene is a gene that codes for a protein that potentially can transfer a normal cell into a malignant cell. Most normal cells undergo a process of cell death- apoptosis when critical functions are altered. An activated oncogene cause a cell designated for apoptosis to survive and proliferate instead. Oncogenes are normally influenced by external factor such as viruses or environmental factor. A proto-oncogene is a normal gene that can become an oncogene due mutation which leads to increase in protein expression, hyperactivity and / or loss of regulation. Proto-oncogenes are often involved in signal transduction and have mitogenic effects. Upon activation, a proto-oncogene or its product becomes a tumor-inducing agent. A number of proto-oncogenes have been associated with malignant. These include RAS, MYC, WNT, ERK, E6, E7, and TRK. MYC gene is implicated in Burkitt’s lymphoma. Some of the proto-oncogenes will be dealt with in subsequent chapters where the pathogenesis of some virus-associated cancers will be discussed.
A proto-oncogene can become activate by:
· Point mutation,
· Amplification
· Translocation to a transcriptionally active site,
· Chimeric gene creation due to chromosomal rearrangements
1.4.3 Tumor suppressor genes: A tumor suppressor gene is a gene that protects a cell from the first step of the cancer process. Mutation is this gene can cause loss or reduction of its function. The cell then progresses to cancer, usually in combination to other factors. The tumor suppressor genes has a number of functions, including:
· Repression of genes that is essential for cell cycle.
· Coupling the cell cycle to DNA damage.
· Initiation of apoptosis.
· Metastasis suppression.
Example of tumor suppressor genes are Retinoblastoma protein (pRB) found in human retinoblastoma and p53 which is encoded by TR53 gene. P53 as a tumor suppressor gene will be discussed in details in chapter 6.
The main weapon that oncoviruses deploy for cancer pathogenesis is persistence. In order to achieve that, they utilize their ability of evading the immune system. The effect of host immunity in the pathogenesis of oncoviruses will be dealt with in chapter 2.
Some hormones are known to be important in the development of cancer by promoting cell proliferation. Insulin-like growth factors and their binding protein play key role in cancer cell proliferation, differentiation, and apoptosis. But do hormones play a role in the pathogenesis of viral-associated cancer? A number of studies have been undertaking to analyze the effect of hormones in the development of viral-associated cancer. A study by Lindström and Hellberg to investigate the expression of leucin-rich repeats and immunoglobin-like domains 3 (LRIG3) in invasive cancer, and cervical intraepithelial neoplasm (CIN) for possible correlation with other tumor markers; to hormones and smoking involving 129 patients with invasive squamos cell carcinoma and 170 biopsies showing high and low grade CIN, or normal epithelium found that in CIN, there was high expression of the tumor suppressors retinoblastoma, p53, and p16, and E-cardherin or low expression of CK10 , correlated to LRIG3 expression. In addition, prosgestogenic contraceptive use correlated with high expression of LRIG. High LRIG expression correlated significantly with the presence of high-risk HPV infection in patients with normal epithelium or CIN. A study by Lauttia et al to analyze the effect of prokineticins in MCC with Merkel cell polyomavirus infection concluded that prokineticins are associated with Merkel cell polyomavirus infection and participate in regulation of the immune response in MCC, and may influence the outcome of MCC patients. The prokineticins family is family of chemokines-like proteins that are highly conserved across species. Data showed that they have influence in great diversity of biological functions and participate in the coordination of complex physiological activities such as feeding, drinking, regulation of circadian rhythm, and hyperalgesis, and have been suggested to be involved in angiogenesis, inflammation, and cancer. However a study found that relationship between HCC and metabolic factors other than diabetes is inconclusive; although another study found that triglyceride levels were inversely associated with subsequent HBV-associated HCC. Hormones like estradiol 2 (E2) are confirmed cofactors for HPV- associated cervical cancer. Most of the studies analyzing the effect of hormones on viral-associated were inconclusive therefore more studies are needed to elucidate these correlation of hormones and viral-associated cancer.
1.4.3 MiRNA and Oncogenesis: Micro (mi) RNAs are small, noncoding, highly stable 22-base pair nucleic acids that was first discovered in the nematode Caenorhaditis elegans. They are mobile and functional genetic elements. miRNA genes play important roles in developmental timing, morphologic changes, cell proliferation and death, hematopoiesis, nervous system control, pancreatic insulin secretions, adipogenesis, oncogenesis, and viral diseases. A typical miRNA gene is made of 5’-terminal monophosphate and 2’,3’-diol at their 3’-terminus; although some modifications have been reported. Unlike mRNA, which serves as a template for DNA translation to protein, miRNA are unique in the sense that they directly regulate this translation. Several hundreds of these oligomers have been discovered. Viruses produce their own set of miRNAs which have been implicated in virus-associated carcinogenesis. The first to be identified as regulatory viral miRNA was miR-S1 in simian virus 40 (SV40) which promotes the recognition and destruction of infected cells by cytotoxic T cells while the first reported trans-regulatory viral miRNA was miR-LAT which targets TGF-β and SMAD3, thereby promoting cellular proliferation and preventing apoptosis in HSV-1 infection. A number of studies have tried elucidating the role of miRNA in viral-associated cancers. In EBV infection, miRNA was first described by Pfeffer et al and later 44 mature miRNAs from precursors miRNA have been discovered. Studies have found that they are encoded in two regions: BART and BHRF1. They show variable expression in cell lines and tumor during viral latency and lytic growth. Furthermore it has been found that EBV miRNA can change host miRNA expression. EBV miRNA have been implicated in regulating host cell proapoptotic proteins BBC3/PUMA and BCL2LII/BIM, viral transcripts and transport as well as immunomodulatory targets. The BHRF1 miRNA has been implicated in the inhibition of apoptosis during initial infection and in cell cycle. BHRF1 miRNA are expressed in all forms of EBV latency and also target mRNA transcript involved in host cell apoptosis and cell cycling. Due to these, it has been suggested that miRNA-regulated post-transcriptional regulation of host mRNA may be vital for virus-mediated host cell malignant transformation. Some interesting miRNA-mRNA interactions have been described. These include miR-BARTs-targeting BALF5, a viral polymerase) and several BART cluster such as miRNAs downregulating the oncogenic late membrane protein (LMP) 1 and miR-BART-22 which target LMP2. BART miRNAs are associated with downregulation of proapoptotic and tumor suppressor cellular targets in NPC such as PUMA, WIF1 and APC. In HIV-associated cancers, it has been shown that HIV-1 miRNA is found in the U3 region of the 3’-LTR and it down-regulates cellular apoptosis antagonizing transcription factor (AATF) gene expression. The AATF interacts with POL II and the tumor suppressor pRB. It has also been reported that AATF is associated with endogenous antagonist of prostrate apopotosis-4 (Par-4) which is reported to be associated with the suppression of Bcl2 gene transcription. HIV-1 miR-HI is likely to activate E2F activity and inhibit apoptosis. This might the co-factor in the carcinogenesis process of HIV-1 infection. In HTLV infection, Pichler et al and Yeung et al reported that miRNA is either directly activated by Tax or associated with HTLV-1 induced cell transformation. In their study, Pichler et al selected a limited number of miRNAs with links to cancer and overexpressed in regulatory T lymphocytes. RT-PCR quantification analysis identified upregulated and repressed miRNAs in cell lines derived from ATL patients, HAM/TSP patients, and HTLV-1 or Tax transformed cells. The upregulated miRNAs were miR-21, miR-24, miR-146a, and miR-158 while the repressed was miR-223. Expression of one of the miRNAs (miR-146a) was directly activated by Tax through the proximal NF-kB site of the MIRNA146A gene promoter. Yeung et al on the other hand profiled 327 human miRNAs in seven HTLV-1 transformed cell lines and four from PBMC samples from acute ATL patients. Among fifteen miRNAs whose expression was consistently modified compared to paired controls, only three : miR-93, miR-130b, and miR-18a were induced upon activation of normal PBMC with phorbol myristate acetate. By using RT-PCR analysis, the differential expression of miR-93 and miR-130b was confirmed by the authors. Furthermore, luciferase reporter assays and computational analysis showed that p53-induced tumor suppressor protein (TP53INP1) was a target which is shared by both miR-93 and miR-130b. Utilizing antagomirs for miR-93 and miR-130b restored the expression of TP53INP1 and increased the apoptosis of HTLV-1 transformed MT4 cells. siRNA knock-down of TP53NPI1 saved MT4 from cell death induced by miR-130b antagomirs. Increased expression of miR-130b was partly associated with transcriptional activation by Tax. A number of miRNAs have been reported in ATL cells. This means HTLV-1 may either subvert or include cellular miRNAs for persistence and transformation. However because of lack of conclusive data, more studies are needed to elucidate the role of miRNAs in HTLV-associated oncogenesis and identify more miRNA for proper diagnosis and management of HTLV- associated diseases. miRNAs are essential component of viral oncogenetic process. Understanding their roles will aid in formulating therapeutic drugs and biomarkers of oncoviral diseases.
1.4.4 Epigenetic: Epigenetic was defined by Robin Holiday as heritable changes in gene expression that do not result from any alteration in the DNA sequence. It includes the methylation status of DNA, and the posttranslational modification of histones. Epigenetic process plays an important function in tumorigenesis in mammals. The best known marker of epigenetic is DNA methylation, which together with specific histone modification and specific miRNA are believed to be a defining molecular landscape that is altered in cancer. DNA methylation occurs mainly on the cytosine that is before guanine resulting in the formation of 5-methlycytosine. These dinucleotide sites are referred to as CpGs. The CpGs are found in the promote regions of about half of all gene. Called the CpG Island because they are distributed asymmetrically into the CpG-poor region and diverse regions. These CpG Island are usually unmethylated in the normal cells while sporadic CpG sites in the rest of the genome are normally methylated. DNA methylation is a normal process in cells of the mammals that allows normal expression pattern to be maintained. It is involved in genomic imprinting, X-chromosome inactivation in females, and silencing parasite as well as foreign elements, among others. But methylation of CpG Islands in the promoter region is associated with gene silencing and aberrant DNA methylation as reported in most cancers leading to the silencing of some tumor suppressor genes. As explained earlier, oncoviral genomes disrupt the host genome by insertion mutations and chromosome rearrangement, predisposing the infected cells to cancer. It is also known that viral genes are associated with aberrant methylation profile in host-specific genes in human cancers. A number of studies have attempted to elucidate the epigenetic changes in the viruses. In EBV, epigenetic regulation of viral gene is essential event in the life cycle of the virus. The expression of latent viral oncogenes, RNA, and miRNA is under epigenetic control by DNA methylation and histone modification resulting in complete silencing of EBV gene. Methylation of EBV genome protects the host cell through the subduing the transforming latency gene. However, the virus uses DNA methylation to maximize its persistence strategies and hide itself from immune detection, inhibiting the expression of viral latency proteins which are recognized by cytotoxic T cells. HPV infection has also been associated with viral and host epigenetic modification involving DNA methylation and histone modification which contribute to pathogenesis and tumorigenesis. Viral DNA methylation is associated with carcinoma than asymptomatic infections or dysplasia. For e.g., HPV 16 and 18, the LCR and E6 sequences were reported to be unmethylated regardless of the stage of neoplastic progression while the L1 region was densely methylated. Methylation studies have shown that in HPV16, the LCR was methylated in some primary cervical carcinoma, especially at E2-binding sites (E2BS). E2BS have been shown to inhibit the binding of E2 and that methylation is related to activation of E6 and E7 viral proteins. Similarly, epigenetic changes have been described in HCV infection. It has been reported that this epigenetic alteration is produced by viral proteins. HBx protein and various HBs envelope proteins are responsible for the alteration of major signaling pathways. HBx is the key factor in initiating epigenetic alteration induced by the virus. HBx interacts with DNMT to initiate the epigenetic alteration. HBV replication is also associated with epigenetic marker such as the acetylation of H3 and H4. Epigenetic alterations have been described in almost all the described oncoviruses. More studies are needed for us to understand the epigenetic process better because it will give us option of developing better and effective chemotherapy.
1.5 Molecular Tools for research in Oncoviral Infections
Advances in molecular techniques have enhanced our current understanding of the carcinogenetic process involved in viral-associated cancer development. A summary of the current molecular tools utilized in viral-associated cancer research are presented.
1.5.1 Polymerase chain reaction (PCR) Assay: Most molecular-based studies begin
with the extraction of DNA from a particular organism, followed by the amplification (i.e., generation of many copies) of particular segments of DNA using the polymerase chain reaction (PCR). The utility of PCR lies in the fact that only minute quantities of DNA are needed (e.g., nanogram amounts). Figure 1 describes the various stages of a typical PCR technique.
Figure 1: The PCR method begins with total genomic DNA extracted from an organism. The DNA is combined with site-specific primers, taq polymerase, and other reagents (e.g., MgCl2, buffer, dNTPs) and subjected to repeated cycles, each of which consists of a denaturation phase, annealing phase and extension phase. Denaturation separates double stranded DNA, allowing primers to anneal to specific sites, followed by incorporation of deoxynucleotide triphosphates (dNTPs; A, C, G, T), thereby extending the target site in the 5'-3' direction (on both separated strands). The first cycle is completed when one round of denaturation, annealing and extension is finished, resulting in two new copies of the target site. Subsequent cycles (typically 30-35) repeat the 3-phase process, resulting in many million-fold copies of amplified DNA.
Recent advances in biotechnology and molecular biology has led to the development of a variety of PCR assay such as real-time PCR, PCR-ELISA, quantitative-PCR, multiplex- PCR, etc which can not only help in identification but also characterization of various pathogens such as oncoviruses.
1.5.2 Markers and Methods
There are many types of genome markers used in molecular virology; these include microsatellite, minisatellite, restriction fragment length polymorphism (RFLPs), and genome sequence data. Markers generated by these methods can also be visualized in different ways. Traditionally, microsatellite and RPLFs are visualized as discrete bands revealed by agarose electrophoresis. The genome analysis can be visualized using polyacrilamide gels and autoradiography. Due to advancement in molecular techniques, these markers can also be visualized using chemiflourescence and genetic analyzer which detect fluorescent emission of a labeled primer or fluorescently- labeled nucleotide of a genome sequence. These markers and visualization methods are by no means comprehensive lists, and the techniques to be utilized depend greatly on the question involved. But understanding different types of information provided by different markers can assist in deciding which is best for a particular study. A marker is a biological indicator that signal change in the physiological state, stress, or injury due to disease or the environment. There are three types of markers. Anonymous markers include those generated by a method called amplification fragment length polymorphism (AFLPs). The technique uses restriction enzymes combined with PCR to generate many unique fragments that can be used to distinguish organisms based on their genomes. The technique involves three steps: 1. Restriction of the DNA and ligation of oligonucleotide adapter, 2. Selective amplification of set of restriction fragments, and 3. Gel analysis of the amplified fragments. With this method, sets of restriction fragment may be visualized by PCR without knowledge of the nucleotide sequence and allows for specific co-amplification of high number of restriction fragments. A disadvantage of AFLPs is they are limited in the type of information they provide. Another similar method is called random amplified polymorphism DNA (RAPD) which just like AFLP generates dominant markers which can be visualized using agarose gel electrophoresis. RAPD uses PCR primer set to randomly amplify DNA fragment scattered throughout the genome. Another class of markers is sequence-tagged site (STS) markers which is a short (200-500bp) DNA sequence that has a single occurrence in the genome whose location and base sequence are known. STS can easily be detected with PCR using specific primers designed by the investigator. One type of STS is simple sequence repeats (SSRs) or variable tandem repeats (VNTRs). Unlike AFLP, a prior knowledge of specific region containing tandemly repeat nucleotide motifs is required. It focuses on microsatellite region of the genome. Most SSRs consist of two or three nucleotide. The number of SSRs within a given microsatellite region of the genome often varies among individuals. The third class of markers used by molecular virologists is Sanger sequencing in which the markers are derived from direct DNA sequencing of the targeted region within the genome. Just like STS, DNA sequencing requires precise knowledge of the specific genes, or genes region that is of interest to the investigator. This technique is ideal for studying evolutionary trend of viruses. DNA sequencing also enables the development of a marker type, single nucleotide polymorphisms (SNPs). When multiple sequence of a particular region is generated for multiple members within a species, a single base difference among individual are often detected. Another approach to targeting individual gene is whole genome sequencing. A new method that is rapidly generates sequences that can be analyzed and compiled into whole genome is next generation sequencing (NGS) which is now becoming an important tool for molecular virologists interested in investigating the entire genome of a particular virus. All the molecular methods have their strengths and weakness; therefore choosing a particular method depends on the research aim.
1.5.3 Experimental Methods: Which would suit my research?
A key question to ask when thinking of utilizing molecular technique in oncoviral research is: which is best suited for my particular question? The answer to this question will be determined by a number of factors, all of which must be evaluated both independently and collectively in order to arrive at a cohesive plan before launching a successful molecular study in oncoviral research. Are you interested in:
1. Characterizing a specific protein or gene involved in the pathogenesis of a particular oncovirus?
2. Do you want to investigate immune response and immunity in viral-associated cancer?
3. Do you want to develop and evaluate vaccine, host-viral interaction, non human primate models?
4. Do you want to evaluate a rapid diagnostic test?
5. Are you interested in constructing evolutionary trend of a particular oncovirus?
There are multiple methods to use in assessing your question but thinking carefully about what you want to achieve will determine to which degree you answer your question and publish it within the field of molecular oncovirology.
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