The Presence and Possible Role of Virus-Host Chimeric Transcripts in Adult T-Cell Leukemia-Lymphoma
Authors: Katsuya Hiroo , Miyazato Paola , Islam Saiful , Tan Benjy Jek Yang , Inada Yuki , Matsuo Misaki , Ueno Takaharu , Tokunaga Masahito , Hata Hiroyuki , Yamagishi Makoto , Fujisawa Jun-Ichi , Watanabe Toshiki , Uchimaru Kaoru , Utsunomiya Atae , Kimura Shinya , Satou Yorifumi 
Published: November 2019

The retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host genome and persists for the lifetime of the host. There are tens of thousands of different infected clones in a HTLV-1 carrier and each clone can be identified by its unique viral integration site. Only about 5% of infected people develop the hematological malignancy, adult T-cell leukemia-lymphoma (ATL). However, it is unclear how a certain infected clone, among various different ones, is selected as a malignant clone. It has been reported that viral integration alters transcripts of the cellular host genes adjacent to the integration site, even generating truncated or virus-host chimeric transcripts. Because each infected clone has a unique viral integration site, each clone possibly has unique virus-host chimeric transcripts, which were not present in the host before infection. Therefore, we hypothesized that the integrated provirus generates virus-host chimeric transcripts that may play a role in the clonal selection of the HTLV-1-infected cell.
We previously reported HTLV-1 DNA-capture-seq using biotinylated DNA-probes for the viral genome, to increase the sensitivity and efficiency of viral-sequences detection. In this study, we used HTLV-1 RNA-capture-seq for PBMCs samples from ATL patients to test the hypothesis in a highly sensitive manner. The results showed the presence of chimeric transcripts in 19 out of 30 ATL patients. We next quantified the abundance of chimeric transcripts by droplet digital PCR, and found that the expression levels of chimeric transcripts were similar to those of viral RNAs containing splice junction of HBZ, in 5 of 19 chimeric transcripts positive ATL cases, although the levels varied among different ATL cases. To identify the whole sequences of the chimeric transcripts, we performed Oxford Nanopore sequencing. This approach revealed that the HTLV-1 provirus generates various splicing chimeric transcripts with the host genes in both viral sense and antisense orientations. The transcriptional start site of most of the sense chimeric transcripts was the R region of the 5'- or 3'-long terminal repeats (LTRs) in the proviral sequences, indicating that the chimeric transcripts were generated using the viral promoters because the LTRs work as a promoter for the viral transcripts. Given the structure of the chimeric transcripts with the viral promotors, the expression of the fused host genes could be enhanced by generating the chimeric transcripts. We evaluated the mRNA expression of the fused host genes of the chimeric transcripts by RNA-seq, and the results correlated with those obtained by ddPCR.
To clarify the impact of viral integration on the clonal expansion, we analyzed HTLV-1-infected Jurkat cells. The clonality analysis of infected cells by HTLV-1 DNA-capture-seq showed that some infected clones were remarkably expanded for 4-6 months culture. We also confirmed that some of them harbored virus-host chimeric transcripts by HTLV-1 RNA-capture-seq.
This study revealed the expression levels and the structures of virus-host chimeric transcripts in ATL patients. We are currently investigating the functional role of chimeric transcripts in the clonal proliferation of infected cells in vitro.

Disclosures
Uchimaru: Daiichi Sankyo Co., Ltd..: Research Funding. Kimura:Novartis: Honoraria, Research Funding; Ohara Pharmaceutical Co.: Research Funding.