Tumor viruses cause cancer; thus, extensive studies are being conducted on them. In this article, we will review the basic medical research on the current clinical genetic tests for tumor viruses such as human papilloma virus, hepatitis B virus, and T-cell leukemia virus. Recently, clinical genetic tests have been developed for quick diagnosis of the tumor virus infection. Additionally, we will review the mutagenesis of murine leukemia retrovirus. In particular, we will focus on the insertional mutagenesis. This will help in deciding the direction of future virus research by combining clinical and basic research.
- tumor virus
- gene analysis
- clinical examination
- cancer diseases
1.1 History of tumor virus research
Tumor virus research is an important subject to understand the molecular biology of tumor viruses and carcinogenesis. Dr. Katsusaburo Yamagiwa, a Japanese pathologist, is famous for the experiment in which coal tar was applied to rabbit ears to induce carcinogenesis in 1915. He was also a candidate for the Nobel Prize in Medicine. The following year, his student Dr. Akira Fujinami was appointed to study carcinogenesis in Germany for 4 years. After returning to Japan, in 1900, he became the first professor at the Department of Pathology, Kyoto Imperial University Medical College. In 1905, he started studying chicken tumors, and as early as 1910, he established a chicken breast tumor transplant system. He had suggested the possibility of carcinogenesis by filterable pathogens, which were later called Fujinami sarcoma virus (FSV). He is also well known for the discovery of Japan schistosomiasis.
Interestingly, in the same year, Peyton Rous, who was studying at the Rockefeller laboratory in the United States, also established the avian sarcoma transplant system. In 1966, Rous received the Nobel Prize in Physiology and Medicine for the discovery of carcinogenic viruses. Hanafusa et al. elucidated the FSV gene structure and identified the similarity with the Rous sarcoma virus gene structure . Both FSV and Rous sarcoma virus belong to the same alpha-retrovirus. FSV has the oncogene v-fps in its genome . It also has long terminal repeats (LTRs) at the ends of the genome that stabilizes as parts of the host cell genome when inserted into host mesenchymal cells after reverse transcription. In addition to the host cell division, the viral genome also replicates during the DNA replication cycle. Tyrosine-protein kinase transforming protein Fps encoded by v-fps has the ability to induce transformation by promoting tyrosine phosphorylation of the PDGFbeta receptor [3, 4]. Thus, the Japanese scientists have greatly contributed to the field during the early days of oncology research.
1.2 Viral genome integration
Retroviral integration is a type of insertional mutagenesis. Integration processes are widely studied from biochemistry, and their molecular mechanisms are elucidated. Retrovirus-induced tumorigenesis is divided into two types. First, the retroviral genome carries the oncogene such as
The retroviral promoter and enhancer elements in LTRs originally promote the expression of retroviral genes such as
To date, extensive research has been conducted on target motifs for retroviral insertion to better understand tumorigenesis, neuronal damage, aging, and immunodeficiency including acquired immunodeficiency syndrome (AIDS). Retroviral-host DNA interactions support specific host DNA structures [16, 17, 18, 19, 20, 21] but are less likely to be inserted into particular base sequences. Previous studies on human immunodeficiency virus (HIV)-1 insertion have shown that the insertion sites are distributed in 10-bp cycles on the nucleosome surface of the outward DNA major groove of chromatin [16, 22].
HPV is one of the most common viruses that infect more than half women once in life through sexual intercourse. It is a small DNA virus with approximately 8000 bp circular double-stranded DNA in its genome. The viral particle has icosahedral capsid structure as its outer shell. The virus has been known to be involved in the development of cervical, anal, and vaginal cancer and condyloma acuminatum. Many types of HPV are known, and high-risk HPV based on this cancer risk is concentrated in specific subgroups epidemiologically. In particular, types 16 and 18 have high carcinogenic risks of cervical intraepithelial neoplasia (CIN) and cancer. Type 16 has been detected in the Japanese 5-year-old girls . High-risk group (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) causes bladder cancer and pharyngeal cancer in addition to vulvar and cervical cancer. Low-risk group (HPV2, 27, and 57) causes verruca vulgaris that develops in the hands and feet .
HPV genome has early genes (E1, E2, E4, E5, E6, and E7) and late genes encoding viral capsid proteins (L1 and L2). E6 and E7 are considered to be involved in carcinogenesis. E6 binds to a tumor-suppressor gene product p53 and degrades p53 through promoting the ubiquitination. It also contributes to carcinogenesis by reactivating telomerase reverse transcriptase (hTERT) and degrading proteins with PDZ domain [shared by—post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and Zonula occludens-1 protein (zo-1)]. E7 binds to the tumor suppressor gene pRb and contributes to carcinogenesis by releasing and activating the transcription factor E2F bound to pRb. It is also involved in carcinogenesis by interacting with cyclin-dependent kinase (Cdk) inhibitors p21 and p27 to evade cell cycle checkpoint mechanisms and apoptosis. However, in many cases, immortalization does not occur only by the actions of human cervix mucosal cells E6 and E7. In the well-known HeLa cell line, telomerase is activated, preventing the gradual shortening of telomeres in cell death.
High-risk HPV can be detected in more than 90% patients suffering from cervical cancer and dysplasia, which is a precancerous lesion, and in the vulva of 5% normal women. Although HPV is also integrated into the genome of host cells, its integration pattern has unique features. The E6 and E7 genes are retained in the provirus with frequent deletion mutations in E2 gene. Thus, the viral genome is also found to be truncated in the complex reaction of integration, but its carcinogenicity is rather enhanced. Low-grade dysplasia of the cervix and cervical intraepithelial neoplasia (CIN) is well known. These pathological changes are reversible and are known to spontaneously disappear within about 3 years. However, about 10% infected patients sustain the infection for more than 3 years, and cervical cancer occurs after several months to several decades in some of them. HPV genome is integrated into the host cell genome during the progression of CIN, and the progression becomes irreversible.
2.1 Clinical test for HPV detection
HPV-DNA typing tests have been conducted using real-time PCR, loop-mediated isothermal amplification (LAMP) [25, 26], invader, and hybridization methods. In recent years, the Clinichip method  combining LAMP is broadly available in advanced countries including Japan due to the ability to amplify genes at a constant temperature. The LAMP method is very specific because it uses four primers to recognize the six regions of the HPV genome. Nonspecific amplification does not occur because the amplification reaction depends on the order of the six regions of the HPV gene. In addition, since the amplification product constitutes the target gene sequence, the target gene can be detected immediately by the presence or absence of amplification.
In an environment where electricity availability is limited, easy-to-use and durable reagents and equipment are desirable. In recent years, attempts have been made to spread virus testing as point-of-care tests (POCT) . For some pathogens such as malarial parasite (
HBV has been identified as the pathogen that causes acute and chronic hepatitis B [29, 30]. HBV is a DNA hepatitis virus and has a double structure of an envelope and a core. In persistent infections, the HBV genome is often inserted into the hepatocyte genome and is not eliminated by the host’s immune system. Several studies on many common insertion sites have identified common integration sites in the HBV genome by full genome sequence of hepatocellular carcinoma . The envelope protein of HBV covers the HBs antigen, and the core protein is called the HBc antigen. HBV DNA encodes HBs antigen, HBc antigen, X protein, and DNA polymerase. The HBe antigen is translated from the pre-C gene and the downstream HBc antigen. It is released into blood as a soluble protein during HBV virus growth and therefore considered a marker for the virus growth. In asymptomatic carriers positive for HBe antigen, the host immune system continuously reacts with HBV-infected hepatocytes, and biopsy reports show persistent lymphocyte infiltration in the portal vascular area of liver tissues. If the damage persists for a long time, remodeling of the hepatocytes occurs and progresses to cirrhosis with obvious fibrotic crosslinks. After all, when seroconversion occurs in HBV carriers negative for HBe antigen and positive for HBe antibody, the patient becomes an inactive carrier. HBV particles remain alive during periods of inactivity. Host immune system can attack infected hepatocytes, and hepatocyte regeneration may occur with genomic mutations, and the effects of the inserted HBV genome persist. Hepatocellular carcinoma can develop with persistent liver dysfunction associated with liver cirrhosis.
3.1 Clinical test for HBV detection
For the diagnosis of hepatitis B, HBs antigen/antibody, HBc antibody, HBe antigen/antibody, and HBV DNA are detected, and HBV DNA polymerase activity is measured. HBs antigen is produced continuously in patients with HBV infection; thus, positive results for HBe antigen can diagnose current infection with HBV. Recently, the HBs antigen measurement system has become very sensitive, and infected people have been found to be positive for HBs. In addition to antigen or antibody detection method, viral DNA detection system (quantitative PCR, LAMP method) has been used for clinical tests. Liver fibrosis is seen as a precancerous stage of HBV and hepatitis C virus (HCV) infection. Liver biopsy and qualitative analysis of hyaluronic acid and type IV collagen in blood are well-known methods to evaluate liver fibrosis progress. Monitoring of glycolysis of serum M2BP (Macingo binding biomarker) is used to evaluate liver fibrotic stage . Recent studies have suggested that the most common cause of nonexclusion of HBV is covalently closed circular DNA (cccDNA). When HBV infects hepatocytes, the incomplete duplex circular DNA genome becomes a complete duplex and is retained in the nucleus in the form of cccDNA. This HBV gene is the source of HBV replication. Since cccDNA is an extremely stable biochemical structure, it can be used as a monitoring marker after the infection. As the nucleic acid analog drug has little effect on reducing HBV cccDNA, a high risk of reactivation remains. If host immunity is suppressed, the reactivation occurs from this cccDNA as a starting transcriptional point. Therefore, elimination or suppression of reactivation will be central to future therapeutic strategies .
In the clinical tests for HBV detection, the detection sensitivity of the enzyme-linked immunosorbent assay (ELISA) method for HBs antigen is higher than those of other tests. Each available method depends on the titer of the used antibody, and the sensitivity varies. In particular, the mutation in the gene encoding the envelope protein HBs antigen may cause false negative results. Many test kits increase sensitivity by using multiple monoclonal antibodies or a polyclonal antibody that has an affinity for multiple epitopes. In some cases, antibodies that recognize epitopes within the viral particle are used instead of surface proteins with many mutations. However, the high sensitivity of many kits may instead cause false positive results. Accordingly, actual early diagnosis requires careful evaluation of test results in combination with other clinical tests.
4. HTLV type 1 (HTLV-1)
Another important human carcinogenic virus is human T-cell leukemia virus type 1 (HTLV-1). This virus has the potential to cause adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy (HAM), and HTLV-1 uveitis (HU). These diseases develop in HTLV-1 carriers after prolonged latent infection. HTLV-1 carriers are particularly prevalent in southwest Japan, including Kyushu, Shikoku, and Okinawa. HTLV-1 was isolated and reported by Gallo R as the first human pathogenic retrovirus in 1980. Yorio Hinuma of Kyoto University in Japan precisely analyzed the genomic structure and identified the HTLV-1 . HTLV-1 is a weakly infective virus particle, and cell-to-cell transmission is the main route of infection.
4.1 Molecular biology of HTLV-1
ATL is a hematologic malignancy in which abnormal lymphocytes appear in peripheral blood and infiltrate various organs throughout the body. Kiyoshi Takatsuki of Kumamoto University in Japan and Taku Uchiyama of Kyoto University collected samples. In 1981, Hinuma identified HTLV-1 to be the causative virus of ATL; this was the first report on tumor-induced retrovirus in humans. Most infected cells are CD4 + CD25 + T cells. Viral receptors are thought to involve other proteins and heparan sulfate. HTLV-1 is an approximately spherical virus particle about 100 nm in diameter and has an envelope. It has an RNA genome composed of the genes common to retroviruses such as
Tax and HBZ may be the carcinogenic potentials of HTLV-1. Tax is a potent transactivator essential for the activation of the viral promoter 5′ LTR. After entering the cell by infection, the virus particle capsid is lost, and the HTLV-1 genomic RNA is reverse transcribed to form genomic duplex DNA. Thereafter, the integrase encoded by the viral genome and the DNA end forms a complex, which contacts the genomic DNA on the cell side, and inserts by catalytic reaction. The 5-base-long sequence of the targeted host cell genome is duplicated at both ends of the inserted viral genome in the insertion process. Tax, the transcript of TLV-1 regulatory gene tax, not only binds to the HTLV-1 promoter to promote viral transcription and expression but also interacts with cell-side transcription factors to activate host gene transcription activity. Therefore,
4.2 Insertion site of HTLV-1
At present, in addition to the carcinogenesis by the proteins of these viruses themselves, attention has been focused on the activation of host genes at the insertion site. The integrated HTLV-1 provirus has the LTR  in both directions for the transcription. This sequence has many motifs that bind to transcription factors on the host and have strong promoter/enhancer activities. Therefore, there may be an increase in constitutive expression of the gene at the insertion site. Ogawa performed systematic genome analysis of ATL cells in 426 ATL patients and showed that genes interacting with Tax, T-cell receptor-NF-κB signal transduction, T-cell transport, and other T-cell-related pathways as well as genes related to immune surveillance are injured by the integration. Also, the expressions of VAV1, IRF4, and FYN related to lymphocyte maturation and signal transduction, chemokine receptors CCR4 and CCR7, and gene fusions (CTLA4-CD28 and ICOS-CD28) were enhanced in the infected cells . Viral transcripts were mainly derived from the antisense strand. Also, the suppression of
4.3 Clinical test for HTLV-1 detection
To date, no chromosomal abnormality specific to HTLV-1 has been observed, and Southern blot or inverse PCR methods are used for diagnosis; however, in recent years, multiplex LAMP (RT-LAMP) using universal probe has been developed. The method detects both HIV and HTLV-1 RNA from the same sample . HAS HTLV-1 analyzing system (HAS)-Flow method has also been proposed to evaluate the progression of ATL stage by applying flow cytometric analysis focusing on surface markers of infected immune cells .
5. Integration model in mice lymphomas
5.1 The specificity of insertion sequence of the retrovirus genome
As experimental models of MLV integration, BXH2 , AKXD [41, 42, 43, 44], and SL/Kh [20, 45, 46, 47, 48, 49, 50, 51, 52] are well-known mouse strains into which endogenous provirus genomes have been inserted. The genome of an endogenous MLV, AKV , was integrated into
To study MLV genome insertion
5.2 Integration target sequence
To date, such significantly selective insertion into the specific gene has not been known. When insertion further occurs in an oncogene (e.g.,
In AKXD and SL/Kh lymphoma genome,
SL/Kh mouse has multiple copies of the endogenous virus, which produces viral particles and infects immature B-cell lineages suitable for tropism. Several common insertion sites such as Stat5a and Stat5b and well-known genes such as c-myc have been identified till date. Interestingly, many of these insertions occur upstream of the translational initiation site of the protein, so that many intact targeted proteins are produced. The fact that many insertions occur in such specific genes suggests that DNA structures such as palindromic sequences are targeted. However, insertions in a short 50-bp segment cannot have such secondary structure. MLV insertion was observed in almost the entire genome of this SL/Kh acute lymphocytic leukemia/lymphoma. The integration target segment is located just before the translation initiation origin of the Zfp521 gene, which was first reported as a retrovirus insertion sequence. As a result of the insertion, downstream Zfp521 gene is constitutively expressed, and the upregulation may be responsible for acute lymphocytic leukemia/lymphoma.
5.3 Interaction partners for MLV integrase and recent developments in the biosafety of gene therapy
Recently, several cellular proteins including transcription regulatory factors and chromatin and RNA-binding proteins have been discovered as potential interaction partners for MLV integrase . The bromodomain and extra-terminal domain (BET)-containing family of proteins (BRD2, BRD3, BRD4, and BRDT) act as cellular-binding partners of MLV integrase and preferentially bind to open chromatin regions that are enriched for transcription start sites, CpG islands, DNase I-hypersensitive sites, and proto-oncogenes [68, 69, 70, 71, 72, 73]. The BET family of proteins plays a critical role in regulating gene transcription through epigenetic interactions between dual bromodomains and acetylated lysines on histones H3 and H4 during cellular proliferation, cell cycle progression, and differentiation processes [8, 9].
The dual bromodomain protein of BRD2 promotes B-cell expansion and plays a critical role in the regulation of mitogenic response genes, particularly cyclin A, in B cells . In addition, it has been shown that the inhibition of BRD4 leads to the suppression of germinal center B-cell differentiation and antibody responses through the down-regulation of nuclear factor-κBp65 activation and B-cell lymphoma 6 expression . Hence, these BET proteins might broadly contribute to tumorigenesis in cooperation with MLV integration in spontaneous B-cell lymphoma development, and these findings likely provide novel standpoints of proto-oncogene function as well as a better understanding of MLV insertional mutagenesis [15, 17].
The stable integration of retroviral vectors is suitable to deliver therapeutic genes into cells to correct genetic diseases. MLV-based retroviral vectors are one of the most frequently used gene delivery vehicles in gene therapy studies for primary immunodeficiencies such as adenosine deaminase deficiency-severe combined immunodeficiency [10, 18, 19]. However, a subset of patients developed serious adverse events including leukemia that could be attributed to the integration pattern and vector design [10, 18]. MLV-based retroviral vectors integrate in the vicinity of transcription start site through the interaction between the C-terminal tail of MLV integrase (amino acids 381–408) and the ET domain of BET family of proteins [18, 20]. To resolve side effects such as leukemia, BET-independent MLV vectors have been developed by truncation of the C-terminal tail of MLV integrase or the single W390A mutation [18, 20]. Most recently, next-generation BET-independent MLV vectors have been engineered with the addition of the chromodomain of Chromobox protein homolog 1 (CBX1) to MLV integrase_W390A. The MLV integrase_W390A-CBX1 efficiently targets integration away from traditional markers of MLV integration including gene regulatory elements . These findings open new avenues to improve the biosafety of gammaretroviral vectors for gene therapy.
We followed the history of viral carcinogenic research in this review. The topic of viral carcinogenesis mentioned here is a case where the viral genome is inserted into the host genome. Because the molecular mechanism of viral carcinogenesis has been elucidated, significant novel technical methods will be developed in the future.
Clinical sequence analyzes gene changes occurring at diseased sites and provides information useful for diagnosis and treatment of diseases based on the results of gene mutations.
In addition to the various nucleic acid analysis methods such as conventional PCR and LAMP, next-generation sequencing and other rapid technological innovation will aid in the measurement and analysis of numerous gene sequences in a short period. Furthermore, these technologies are expected to be used in the future to analyze infectious diseases such as cancer virus types, HPV high-risk groups, and virus insertion sites. Information on cancer virus infection will also be valuable for designing personalized drugs for cancer patients in the future.
This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (no. 15H05721) and a Grant-in-Aid from the Scientific Research on Innovative Areas, MEXT, Japan (no. P2013-201).
Conflicts of interest
The authors declare no conflicts of interest.
|HPV||human papilloma virus|
|HBV||hepatitis B virus|
|HTLV-1||human T-cell leukemia virus type 1|
|MLV||murine leukemia virus|