**5. Integration model in mice lymphomas**

## **5.1 The specificity of insertion sequence of the retrovirus genome**

As experimental models of MLV integration, BXH2 [40], AKXD [41–44], and SL/Kh [20, 45–52] are well-known mouse strains into which endogenous provirus

**41**

develops.

transcription.

**5.2 Integration target sequence**

*Basic Research and Clinical Examination of Tumor Virus*

genomes have been inserted. The genome of an endogenous MLV, AKV [14], was integrated into *gltscr 1* [20], which has been characterized as a tumor suppressor gene. This locus was identified as *emv11* on chr 7. In our previous work, the inserted AKV genome was shown to contain the full-length MLV genome and expressed to form a retroviral particle, which had the ability to infect lymphocytes according to its retroviral tropism. In the consequent tumorigenesis, the phenotype of the murine tumor is generally variable in inbred strains. For example, AKR/J mice are susceptible to T-cell lymphoma/leukemia [53–55]; however, in BXH2 mice [39, 40], tumors resemble myeloid and lymphocytic tumors. Even if the MLV is integrated into identical genes, the tumor phenotypes are variable [56]. It is likely that the individual genetic backgrounds of the inbred mouse strains determine the phenotype in combination with the integration-induced expression of oncogenes. Thus, oncogenes are responsible for the tumorigenesis; however, the phenotypes of the tumors are determined by other elements, including the genetic background. The insertion site of MLV has been studied using model mouse strains. SL/Kh strain is known to be a model of acute lymphocytic leukemia/lymphoma by MLV infection, and the target sequence of integration has been identified. This phenomenon may not always be generalized in human retroviruses as MLV LTR is very long and has palindromic motifs unlike HTLV-1 LTR. This sequence motif may contribute to the stabilization of the structure as the tetrameric integrase binds to the LTR. Such a stabilizing structure is not found in HTLV-1, and the retroviral genome may differ in HTLV-1 and MLV in this regard. The existence of a specific sequence motif in the genome insertion of the virus has been the subject of much debate and was initially resolved negatively in early days. The conclusion was that a virus integration is a random event [57] (by Howard Temin, who discovered reverse transcriptase and won the Nobel Prize in 1975). The randomness was proved through the genome analysis of infecting fibroblasts with integrated MLV in the culture state artificially. No common sequence motif for the insertion site was found in this way. However, there has been a report that active transcriptional genes can be the target of integration [21]. Besides, there are reports stating that palindrome sequence is favored by integration [16, 58]; however, the target sequence itself is yet to be identified. The idea is that viral insertion affects the transcriptional activity of the host cell genome by the three-dimensional structure of chromatin. A model may be considered in which the integrase complex binds to the site where DNA is locally released during

To study MLV genome insertion *in vivo,* inbred strains of mice harboring many copies of endogenous MLV genomes were developed. SL/Kh is one of the inbred strains (Tsukuba, Japan; http://www.riken.jp/engn/index.html) [14, 45]. In this strain, the harbored MLV genome is spontaneously and selectively integrated into the genome of the B-cell genome DNA. In reference to the data about MLV insertion sites in the genome of SL/Kh mice, whether MLV insertion occurred preferentially into the specific sequence motifs in the host cell genome was investigated. *Stat5a* [20, 51, 59, 60] and *c-myc* [47] were the target genes for MLV insertion. More than 95% studied SL/Kh mice had such the MLV genome (AKV) insertion in zinc finger protein 521 gene (*Zfp521*) in

To date, such significantly selective insertion into the specific gene has not been known. When insertion further occurs in an oncogene (e.g., *Stat5a* [51] or *c-myc* [47]) in addition to *Zfp521* in the genome of SL/Kh mice, B lymphocytic leukemia

the genomic DNA of B-cell lineage at 7 weeks of age [61] (**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.89331*

#### *Basic Research and Clinical Examination of Tumor Virus DOI: http://dx.doi.org/10.5772/intechopen.89331*

*Viruses and Viral Infections in Developing Countries*

**4.2 Insertion site of HTLV-1**

**4.3 Clinical test for HTLV-1 detection**

on surface markers of infected immune cells [38].

**5. Integration model in mice lymphomas**

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, *tax* is implicated in the early stages of tumorigenesis of infected cells. However, in peripheral blood of most ATL patients, Tax is suppressed by its promoter 5′ LTR deletion and epigenetic modification. Thus, Tax is not essential, at least at the final stage of carcinogenesis. Recently, Matsuoka has suggested that Tax is constitutively expressed in a small fraction of leukemia cells, and this fraction triggers an anti-apoptotic mechanism to establish sustained infection and induces oncogenesis [36]. Tax is induced by a variety of cytotoxic stresses and also promotes HTLV-1 replication. Thus, it protects infected cells from apoptosis and increases the chance of viral infection at the critical phase of disease in the carrier. Another HBZ gene is expressed in all ATL patients causing and promoting

the growth of ATL cells; therefore, it may be the gene responsible for ATL.

not been known to be likely to occur in any particular sequence motif.

**5.1 The specificity of insertion sequence of the retrovirus genome**

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 [39]. 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

As experimental models of MLV integration, BXH2 [40], AKXD [41–44], and SL/Kh [20, 45–52] are well-known mouse strains into which endogenous provirus

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 [35] 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 [37]. Viral transcripts were mainly derived from the antisense strand. Also, the suppression of *Tax* expression and the constitutive expression of *HBZ* were observed in almost all patients. These insertion sites have

**40**

genomes have been inserted. The genome of an endogenous MLV, AKV [14], was integrated into *gltscr 1* [20], which has been characterized as a tumor suppressor gene. This locus was identified as *emv11* on chr 7. In our previous work, the inserted AKV genome was shown to contain the full-length MLV genome and expressed to form a retroviral particle, which had the ability to infect lymphocytes according to its retroviral tropism. In the consequent tumorigenesis, the phenotype of the murine tumor is generally variable in inbred strains. For example, AKR/J mice are susceptible to T-cell lymphoma/leukemia [53–55]; however, in BXH2 mice [39, 40], tumors resemble myeloid and lymphocytic tumors. Even if the MLV is integrated into identical genes, the tumor phenotypes are variable [56]. It is likely that the individual genetic backgrounds of the inbred mouse strains determine the phenotype in combination with the integration-induced expression of oncogenes. Thus, oncogenes are responsible for the tumorigenesis; however, the phenotypes of the tumors are determined by other elements, including the genetic background. The insertion site of MLV has been studied using model mouse strains. SL/Kh strain is known to be a model of acute lymphocytic leukemia/lymphoma by MLV infection, and the target sequence of integration has been identified. This phenomenon may not always be generalized in human retroviruses as MLV LTR is very long and has palindromic motifs unlike HTLV-1 LTR. This sequence motif may contribute to the stabilization of the structure as the tetrameric integrase binds to the LTR. Such a stabilizing structure is not found in HTLV-1, and the retroviral genome may differ in HTLV-1 and MLV in this regard. The existence of a specific sequence motif in the genome insertion of the virus has been the subject of much debate and was initially resolved negatively in early days. The conclusion was that a virus integration is a random event [57] (by Howard Temin, who discovered reverse transcriptase and won the Nobel Prize in 1975). The randomness was proved through the genome analysis of infecting fibroblasts with integrated MLV in the culture state artificially. No common sequence motif for the insertion site was found in this way. However, there has been a report that active transcriptional genes can be the target of integration [21]. Besides, there are reports stating that palindrome sequence is favored by integration [16, 58]; however, the target sequence itself is yet to be identified. The idea is that viral insertion affects the transcriptional activity of the host cell genome by the three-dimensional structure of chromatin. A model may be considered in which the integrase complex binds to the site where DNA is locally released during transcription.

To study MLV genome insertion *in vivo,* inbred strains of mice harboring many copies of endogenous MLV genomes were developed. SL/Kh is one of the inbred strains (Tsukuba, Japan; http://www.riken.jp/engn/index.html) [14, 45]. In this strain, the harbored MLV genome is spontaneously and selectively integrated into the genome of the B-cell genome DNA. In reference to the data about MLV insertion sites in the genome of SL/Kh mice, whether MLV insertion occurred preferentially into the specific sequence motifs in the host cell genome was investigated. *Stat5a* [20, 51, 59, 60] and *c-myc* [47] were the target genes for MLV insertion. More than 95% studied SL/Kh mice had such the MLV genome (AKV) insertion in zinc finger protein 521 gene (*Zfp521*) in the genomic DNA of B-cell lineage at 7 weeks of age [61] (**Figure 2**).

#### **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., *Stat5a* [51] or *c-myc* [47]) in addition to *Zfp521* in the genome of SL/Kh mice, B lymphocytic leukemia develops.

#### **Figure 2.**

*Murine leukemia retrovirus (MLV) integration into oncogenes in SL/Kh lymphoma. In most cases of lymphoma, MLV integrations into more than one oncogenes, including Stat5a, c-myc, Fiz1, Hipk2, and others, are observed. It is not clear why the tumor cells are restricted to pre-B lymphoma phenotype. To date, genetic background dependent on Bomb1 is critical for conferring the phenotype. In addition, Stat5a and Zfp521 genes cooperate with IL-7 and pre-B cell receptor signal pathways, respectively. Host cell phenotype at the integration may contribute to the phenotypes. Differentiation stage of the host cell at the mutagenesis may be one of the critical factors of the phenotype.*

In AKXD and SL/Kh lymphoma genome, *Zfp521* is one of the most frequent targets of MLV integration [62]. This gene locus was first reported as a common integration site, ecotropic retrovirus integration site 3 (*Evi3*) [61–64]. In more than 400 male SL/Kh mice, >90% pre-B-lymphoma genomes acquired the integrated proviral genome within a 50-bp segment located in the region of the second to third exon of *Zfp521*. This target gene carries alternating palindrome sequences [16], which was frequently targeted by MLV insertion. *Zfp521* is one the novel identified target genes. It probably contributes to chondrocyte development [65], in addition to B-cell development that requires an abnormal chimeric gene [64]. Each insertion occurs once or twice during the development of lymphoma within a 50-bp segment located in the second intron to the third exon of *Zfp521*. Most commonly, the insertion occurs at the ":" site in the sequence "CTGAATTG*AAAC:* AACTTCAGC*TGTTT*," where the pair of underlined sequences and the pair of sequences in italics are palindromic sequences [61–63, 66]. The insertion does not occur randomly; they frequently occur at approximately 10-bp intervals and are symmetrically distributed within the shown 50-bp segment (**Figure 3**).

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

**43**

mutagenesis [15, 17].

*Basic Research and Clinical Examination of Tumor Virus*

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.

*Alternate palindrome sequence in Zfp521. The upper and lower sequences represent the native sequence of host murine Zfp521 gene and the junction sequence of host murine Zfp521 gene with the integrated MLV genome, respectively. Green and red letters represent pairs of alternating palindromes. The sequences in the same colors* 

Recently, several cellular proteins including transcription regulatory factors and chromatin and RNA-binding proteins have been discovered as potential interaction partners for MLV integrase [67]. 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 [68–73]. 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]. *In vitro* interaction analysis in human cells revealed that the ET domain of BRDs, BRD4 in particular, interacts with a motif in the unstructured C-terminal tail of MLV integrase, and these interactions are crucial for the recruitment of the MLV PIC to host DNA during integration [10, 11]. Through the recognition of open chromatin structure, BET proteins have been suggested to contribute to the tethering of the MLV PIC to the host chromatin [10, 12]. Furthermore, it has been shown that the cell-permeable small molecule JQ-1, a pan-BET bromodomain inhibitor, prevents the BRD4-acetylated lysine interaction by competitively binding to BRD4 and reduces MLV integration frequencies at transcription start sites [4, 13, 14]. These findings suggest that BET proteins navigate the MLV genome and promote efficient MLV integration around transcription start sites associated with a chromatin structure characteristic of an open, transcriptionally active domain [3]. 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 [15]. 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 [16]. 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

**5.3 Interaction partners for MLV integrase and recent developments in the** 

*are palindromes. The downward arrows represent the hot spot of the MLV integration.*

*DOI: http://dx.doi.org/10.5772/intechopen.89331*

**biosafety of gene therapy**

**Figure 3.**

**Figure 3.**

*Viruses and Viral Infections in Developing Countries*

In AKXD and SL/Kh lymphoma genome, *Zfp521* is one of the most frequent targets of MLV integration [62]. This gene locus was first reported as a common integration site, ecotropic retrovirus integration site 3 (*Evi3*) [61–64]. In more than 400 male SL/Kh mice, >90% pre-B-lymphoma genomes acquired the integrated proviral genome within a 50-bp segment located in the region of the second to third exon of *Zfp521*. This target gene carries alternating palindrome sequences [16], which was frequently targeted by MLV insertion. *Zfp521* is one the novel identified target genes. It probably contributes to chondrocyte development [65], in addition to B-cell development that requires an abnormal chimeric gene [64]. Each insertion occurs once or twice during the development of lymphoma within a 50-bp segment located in the second intron to the third exon of *Zfp521*. Most commonly, the insertion occurs at the ":" site in the sequence "CTGAATTG*AAAC:* AACTTCAGC*TGTTT*," where the pair of underlined sequences and the pair of sequences in italics are palindromic sequences [61–63, 66]. The insertion does not occur randomly; they frequently occur at approximately 10-bp intervals and are

*Murine leukemia retrovirus (MLV) integration into oncogenes in SL/Kh lymphoma. In most cases of lymphoma, MLV integrations into more than one oncogenes, including Stat5a, c-myc, Fiz1, Hipk2, and others, are observed. It is not clear why the tumor cells are restricted to pre-B lymphoma phenotype. To date, genetic background dependent on Bomb1 is critical for conferring the phenotype. In addition, Stat5a and Zfp521 genes cooperate with IL-7 and pre-B cell receptor signal pathways, respectively. Host cell phenotype at the integration may contribute to the phenotypes. Differentiation stage of the host cell at the mutagenesis may be one of the* 

symmetrically distributed within the shown 50-bp segment (**Figure 3**).

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

**42**

**Figure 2.**

*critical factors of the phenotype.*

*Alternate palindrome sequence in Zfp521. The upper and lower sequences represent the native sequence of host murine Zfp521 gene and the junction sequence of host murine Zfp521 gene with the integrated MLV genome, respectively. Green and red letters represent pairs of alternating palindromes. The sequences in the same colors are palindromes. The downward arrows represent the hot spot of the MLV integration.*

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 [67]. 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 [68–73]. 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]. *In vitro* interaction analysis in human cells revealed that the ET domain of BRDs, BRD4 in particular, interacts with a motif in the unstructured C-terminal tail of MLV integrase, and these interactions are crucial for the recruitment of the MLV PIC to host DNA during integration [10, 11]. Through the recognition of open chromatin structure, BET proteins have been suggested to contribute to the tethering of the MLV PIC to the host chromatin [10, 12]. Furthermore, it has been shown that the cell-permeable small molecule JQ-1, a pan-BET bromodomain inhibitor, prevents the BRD4-acetylated lysine interaction by competitively binding to BRD4 and reduces MLV integration frequencies at transcription start sites [4, 13, 14]. These findings suggest that BET proteins navigate the MLV genome and promote efficient MLV integration around transcription start sites associated with a chromatin structure characteristic of an open, transcriptionally active domain [3].

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 [15]. 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 [16]. 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 [10]. These findings open new avenues to improve the biosafety of gammaretroviral vectors for gene therapy.

### **6. Summary**

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.

### **Acknowledgements**

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 Grantin-Aid from the Scientific Research on Innovative Areas, MEXT, Japan (no. P2013-201).

**45**

**Author details**

Tatsuaki Tsuruyama\*, Shuichiro Ogawa and Norishige Yamada

\*Address all correspondence to: tsuruyam@kuhp.kyoto-u.ac.jp

University Hospital, Sakyo-ku, Kyoto, Japan

provided the original work is properly cited.

Department of Drug and Discovery Medicine, Clinical Bioresource Center of Kyoto

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Basic Research and Clinical Examination of Tumor Virus*

HTLV-1 human T-cell leukemia virus type 1

*DOI: http://dx.doi.org/10.5772/intechopen.89331*

HPV human papilloma virus HBV hepatitis B virus

MLV murine leukemia virus

**Abbreviations**

#### **Conflicts of interest**

The authors declare no conflicts of interest.

*Basic Research and Clinical Examination of Tumor Virus DOI: http://dx.doi.org/10.5772/intechopen.89331*
