1.Introduction

Human T-cell leukaemia virus types 1 (HTLV-1) and bovine leukaemia virus (BLV) are closely related retroviruses which can develop leukaemia. Both viruses encode regulatory proteins involved in viral infection and regulation of viral expression, such as Tax, which was originally identified as transcriptional transactivator of viral expression. In HTLV-1 and BLV, Tax are thought to be involved in cellular processes that include cell growth and proliferation, transformation and immortalization, cell cycle progression, apoptosis, cellular DNA repair and cell survival. HTLV-1 Tax is known to modulate the expression of many cellular genes that are related to regulation of cell growth, but little is known about BLV Tax.

Aside from the difference in host range, a notable difference between the two viruses is that infection by BLV is associated with malignancy of B cells, whereas HTLV affects T cells. In this study, to gain a better understanding of the contribution of Tax to HTLV-1- and BLV-induced pathogeneses, genes that play a role in the cascade of signal events regulated by HTLV-1 or BLV Tax were identified in HeLa cells by microarray-based gene expression analysis; and, the regulation of cell cycle and apoptosis by HTLV-1 Tax was visualized by time-lapse imaging.

2.Results

2-1 Host factors regulated by HTLV-1 Tax

Studies about the retroviral protein HTLV-1 Tax have been focused on the function of Tax in regulation of cell cycle and cell proliferation. Expression of Tax has been previously shown to regulate cell cycle progression and apoptosis both positively and negatively, while the molecular mechanisms underlying the regulation of these processes by HTLV-1 Tax remains obscure.

2-1-1 Expression profiling of cellular genes following expression of HTLV-1 Tax

To first know the biological characteristics of HTLV Tax, expression vector encoding Tax was constructed and transfected into HeLa cells. I found that Tax can induce cell cycle arrest at the G1 phase in HeLa cells by flow cytometry analysis and by analysis of the phosphorylation status of Retinoblastoma. Moreover, analysis of Annexin V staining and caspase-3 activity clearly demonstrated that Tax promotes apoptosis.

To analyze the transcriptional effects of HTLV-1 Tax on global gene expression, I performed microarray analysis in HeLa cells and identified 342 genes (269 were up-regulated and 73 were down-regulated) showing differential regulation by >2 fold. The up-regulated genes were clustered within functional groups involved in transcription/translation/RNA processing, signal transduction, immune response, apoptosis, cell cycle and cell growth/proliferation. In particular, a number of molecules involved in immune response were significantly down-regulated by Tax.

I next focused on the mechanism by which Tax induces cell cycle arrest and apoptosis. I identified 17 Tax-dependent genes related to cell cycle regulation resulting in >2.0 fold up- or down regulation (Fig. 1A) and they were involved in response to stress and DNA damage, cell proliferation, mitotic cell cycle and inflammatory and immune response, such as SMAD3, JUN, GADD45β, DUSP1 and IL8. Additionally, 23 pro- and anti-apoptotic genes were deregulated by Tax, including TNFAIP3, TNFRS9, BIRC3 and IL6 factors (Fig 1B). Moreover, microarray results were confirmed by performing qRT-PCR on five up-regulated genes.

2-1-2 Visualizing dynamics of cell cycle and apoptosis regulations by Tax protein.

To clarify whether Tax causes apoptosis independently of its ability to induce G1 arrest, the spatiotemporal patterns of cell cycle regulation in response to Tax expression were visualized in HeLa/fluorescent ubiquitination-based cell cycle indicator (Fucci) cells which allows for dual-color imaging and can be used to distinguish between live cells in the G1 and the S/G2/M phases. I monitored that Tax-induced cell death after cell cycle arrest at G1 phase (Fig. 2A). A drastic increase was observed in the proportion of Tax-expressing cells at G1 phase, as compared with control cells (Fig. 2B). Interestingly, as shown in Figure 2C, overexpression of Tax seemed to reduce the number of HeLa/Fucci2 cells in culture.

2-2 Host factors regulated by BLV Tax

Our group have been previously identified a super-activator form of BLV-Tax (TaxD247G) and an attenuated BLV-Tax (TaxS240P) that exhibit impaired transactivation activity. However, the effects of these mutations on functions other than transcriptional activation are unknown. Therefore, I performed a comparative analysis to identify the functional differences between wild-type and mutants BLV-Tax.

2-2-1 Expression of BLV-Tax and its derivatives in HeLa cells

The expression vectors encoding TaxWT, and mutants TaxS240P and TaxD247G were constructed and transfected into HeLa cells. The protein expression and localization did not differ by any BLV Tax (Fig. 3A-B). The transactivation capacity of Tax showed that TaxD247G induced highest viral LTR-directed transcriptional activation and in contrast TaxS240P displayed markedly reduced activity (Fig. 3C).

2-2-2 Large-scale expression profiling of cellular genes following expression of wild-type BLV-Tax and its derivatives

To analyze the transcriptional effects of BLV Tax on global gene expression, we performed microarray analysis (Fig. 4A) and identified a total of 122, 118 and 139 genes showing differential regulation by >2 fold upon TaxWT, TaxS240P and TaxD247G expression respectively (p<0.05) (Fig. 4B). The genes up-regulated were involved in transcription, signal transduction, cell proliferation, apoptosis, cell adhesion, etc. Interestingly, TaxD247G deregulated more genes belonging to the transcription group; by contrast to TaxS240P which displayed the lowest impact on cellular gene expression. In particular, a number of molecules involved in immune response were significantly down-regulated by all Tax (Fig. 4C).

2-2-3 Validation of expression of host cellular genes induced by BLV-Tax

First, qRT-PCR was used to corroborate the fold change obtained from microarray analysis of the up-regulated genes in the categories of transcription/translation/RNA processing, signal transduction, immune response and regulation of cell growth/cell proliferation; confirming that TaxD247G was inducing a strong response while TaxS240P was inducing lower levels of expression than others Tax (Fig. 5A). I also validated the results of the down-regulated genes which correlated perfectly with the microarray analysis (Fig. 5B). Finally, we utilized Western blotting to determine the levels of proteins (Fig. 5C). In general, results were in good agreement with, and support, the findings of our microarray analysis of HeLa cells.

3.Discussion

In the case of HTLV-1 Tax study, I have visualized that Tax arrested cells at the G1 phase of the cell cycle, thereby inducing apoptosis in HeLa cells and demonstrated that Tax mediates cellular factors with respect to cell cycling and pro- and anti-apoptosis as well as inflammatory and cancer diseases. To our knowledge, this study is the first to highlight the morphological dynamics of Tax-induced cell death after cell cycle arrest at the G1 phase.

Using interesting variants forms of BLV Tax, with elevated (TaxD247G) or reduced (TaxS240P) transactivation activity on the replication and propagation of BLV; I identified cellular genes, which were importantly involved mainly in transcription, signal transduction, cell growth, stress response and immune response. Interestingly, there were differences in the cellular gene expression between the three Tax variants, with emphasis in BLV-TaxD247G which had the ability to induce large numbers of host genes to high levels, contrary to BLV-TaxS240P which induced less numbers of genes and only to relatively low levels. These results suggest the correlation of gene expression deregulation with the transactivation properties of these BLV-Tax alleles. My study also shown the discovery that BLV-Tax and its derivatives profoundly down-regulated the expression of genes involved in the innate immune response.

This overview can be extended into Tax-mediated signalling and further study of the interactions of Tax and cellular factors will provide insights into the mechanisms by which HTLV-1 and BLV Tax can regulate specific cell behaviours.

4.Conclusions

HLTV-1 Tax induced cell cycle arrest at G1 phase and undergone to apoptosis and these events where in correlation with the expression of genes involved in both functions.

BLV Tax variants deregulated the expression of cellular genes, which were importantly involved mainly in transcription, signal transduction, immune response and cell proliferation.

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本論文は2章からなり、第1章は成人T細胞白血病ウイルス1型(HTLV-1)、第2章は牛白血病ウイルス(BLV)の転写活性化因子Taxよって制御される宿主因子について述べられている。

成人T細胞白血病(ATL)を誘発するHTLV-1および地方病性牛白血病(EBL)を誘発するBLVの転写活性化因子Taxは自身のlong terminal repeat (LTR)に作用してウイルス遺伝子の転写を活性化すると同時に、細胞側因子に作用してアポトーシスの抑制、細胞増殖や不死化の誘導、細胞周期の進行および遺伝子変異を誘導し、発癌の引き金を引くと考えられている。一方で、Taxがアポトーシス誘導能や細胞周期停止能を介して細胞増殖を負に制御することも報告されている。本研究では、マイクロアレイによりTaxにより発現が変化する遺伝子を網羅的に解析すると同時に、Tax発現細胞をリアルタイムイメージングにより観察した。

第1章では、HTLV-1 Taxが誘導する細胞異常を解析した。HeLa細胞に構築したFlag融合Tax発現ベクターを導入したところ、Taxのトランスフォーミング活性とは反対の細胞増殖を負に制御する細胞周期のG1期停止およびアポトーシスが誘導された。この興味深いTaxの機能に関連する遺伝子発現の変化をマイクロアレイにより解析した。Tax発現ベクター導入30時間後にRNAを抽出して、18,400個のヒト遺伝子を含むHuman Genome U133 plus 2.0 arrayチップを使用して、発現が2倍以上上昇した188個の遺伝子と低下した61個の遺伝子を同定した。この中に細胞周期関連遺伝子が17個、そしてアポトーシス関連遺伝子が23個含まれていた。マイクロアレイにより得られた結果の再現性をリアルタイムPCRにより確認した。細胞周期関連遺伝子ではSMAD3, JUN, GADD45β, DUSP1 およびIL-8の5遺伝子、アポトーシス関連遺伝子ではNFKBIA, NR4A1, IER3, TNFAIP3, BTG2, TNFRS9, BIRC3 およびIL6の8遺伝子の再現性が確認された。

続いて、Taxが誘導する細胞周期のG1期停止とアポトーシスの関連性を明確にするために、細胞周期可視化プローブ"Fucci"発現細胞に構築したcyan fluorescence protein (CFP)蛍光タンパク質融合Tax発現ベクターを導入して24時間後からリアルタイムイメージングを行った。その結果、Taxが細胞周期をG1期で停止した後にアポトーシスを誘導する過程を経時的に観察することに始めて成功した。

第2章では、BLV Tax による遺伝子発現解析を網羅的に行った。当研究室の先行研究において見出された、BLV LTRに対して高い転写活性化を有するD247G変異型、転写活性化能を消失したS240P変異型および野性型Taxを組み込んだ発現ベクターを構築して、これらのTaxの転写活性化能の違いと関連する宿主因子をマイクロアレイにより解析した。発現が2倍以上変化した遺伝子は、野性型で122個、S240Pで118個、D247Gで139個であった。野生型およびD247では発現上昇を示した遺伝子数が多かったが、S240Pでは発現低下遺伝子数が発現上昇遺伝子数とほぼ同じであった。発現上昇遺伝子には、転写活性化、細胞増殖、細胞周期制御、細胞輸送、リン酸化、アポトーシスおよびストレス機能に関連する遺伝子が含まれていた。発現低下遺伝子には、免疫応答に関連する遺伝子が最も多かった。

マイクロアレイ解析により得られた結果の再現性をHeLa細胞とウシ23CLN細胞で確認した。発現が上昇した遺伝子として転写活性化に関与するFOS、JUN、NR4A2、RORA、GEM、RGS2およびRRAD、免疫応答に関与するTNFAIP6、細胞増殖に関与するCYR61、およびアポトーシスに関与するIER3、TNFRSF12AおよびTNFRSF21を、発現が低下した遺伝子として転写活性化に関与するID2、インターフェロン関連遺伝子IFIT1およびIFIT3、アポトーシス関連遺伝子であるTNFSF10を選択し、リアルタイムPCRおよびウエスタンブロット解析を行ったところ、ヒトおよびウシ細胞の両者においてほぼ良好な再現性が確認された。

マイクロアレイ解析により、BLV Taxが転写活性化および細胞増殖に加えて、これまで明らかにされていなかったストレス応答や免疫応答に寄与することを初めて明らかにした。

なお、本論文は、武田英里、間陽子との共同研究であるが、論文提出者が主体となって解析および検証を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(生命科学)の学位を授与できると認める。