HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown that hbz mRNA promotes T-cell proliferation. In our current studies, we evaluated the role of hbz mRNA on HTLV-1-mediated immortalization in vitro as well as in vivo persistence and disease development. We generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cells in vitro. Viral persistence and disease development were also evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Altered hbz mRNA secondary structure, or loss of hbz mRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1 in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesis in vivo.

Production of Humanized Mice through Stem Cell Transfer.

The development of "humanized" mice has become a prominent tool for translational animal studies of human diseases. Immunodeficient mice can be humanized by injections of human umbilical cord stem cells. The engraftment of these cells and their development into human lymphocytes has been made possible by the development of novel severely immunodeficient mouse strains. Proven protocols for the generation and analysis of humanized mice in the NSG mouse background are presented here. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human umbilical stem cell engraftment of neonatal immunodeficient mice Basic Protocol 2: Human umbilical stem cell engraftment of 4-week-old immunodeficient mice Support Protocol 1: Preparation of human umbilical stem cells Support Protocol 2: Submandibular blood collection from humanized mice and analysis of peripheral blood via flow cytometry.

The PRMT5 inhibitor EPZ015666 is effective against HTLV-1-transformed T-cell lines in vitro and in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) is the infectious cause of adult T-cell leukemia/lymphoma (ATL), an extremely aggressive and fatal malignancy of CD4+ T-cells. Due to the chemotherapy-resistance of ATL and the absence of long-term therapy regimens currently available for ATL patients, there is an urgent need to characterize novel therapeutic targets against this disease. Protein arginine methyltransferase 5 (PRMT5) is a type II PRMT enzyme that is directly involved in the pathogenesis of multiple different lymphomas through the transcriptional regulation of relevant oncogenes. Recently, our group identified that PRMT5 is overexpressed in HTLV-1-transformed T-cell lines, during the HTLV-1-mediated T-cell immortalization process, and in ATL patient samples. The objective of this study was to determine the importance of PRMT5 on HTLV-1 infected cell viability, T-cell transformation, and ultimately disease induction. Inhibition of PRMT5 enzymatic activity with a commercially available small molecule inhibitor (EPZ015666) resulted in selective in vitro toxicity of actively proliferating and transformed T-cells. EPZ015666-treatment resulted in a dose-dependent increase in apoptosis in HTLV-1-transformed and ATL-derived cell lines compared to uninfected Jurkat T-cells. Using a co-culture model of infection and immortalization, we found that EPZ015666 is capable of blocking HTLV-1-mediated T-cell immortalization in vitro, indicating that PRMT5 enzymatic activity is essential for the HTLV-1 T-cell transformation process. Administration of EPZ015666 in both NSG xenograft and HTLV-1-infected humanized immune system (HIS) mice significantly improved survival outcomes. The cumulative findings of this study demonstrate that the epigenetic regulator PRMT5 is critical for the survival, transformation, and pathogenesis of HTLV-1, illustrating the value of this cellular enzyme as a potential therapeutic target for the treatment of ATL.

HTLV-1 intragenic viral enhancer influences immortalization phenotype in vitro, but is dispensable for persistence and disease development in animal models.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and chronic neurological disease. The disparity between silenced sense transcription versus constitutively active antisense (Hbz) transcription from the integrated provirus is not fully understood. The presence of an internal viral enhancer has recently been discovered in the Tax gene near the 3' long terminal repeat (LTR) of HTLV-1. In vitro, this enhancer has been shown to bind SRF and ELK-1 host transcription factors, maintain chromatin openness and viral gene transcription, and induce aberrant host gene transcription near viral integration sites. However, the function of the viral enhancer in the context of early HTLV-1 infection events remains unknown. In this study, we generated a mutant Enhancer virus (mEnhancer) and evaluated its effects on HTLV-1-mediated in vitro immortalization, establishment of persistent infection with an in vivo rabbit model, and disease development in a humanized immune system (HIS) mouse model. The mEnhancer virus was able to establish persistent infection in rabbits, and there were no significant differences in proviral load or HTLV-1-specific antibody responses over a 25-week study. However, rabbits infected with the mEnhancer virus had significantly decreased sense and antisense viral gene expression at 12-weeks post-infection. HIS mice infected with wt or mEnhancer virus showed similar disease progression, proviral load, and viral gene expression. While mEnhancer virus was able to sufficiently immortalize primary T-lymphocytes in cell culture, the immortalized cells had an altered phenotype (CD8+ T-cells), decreased proviral load, decreased sense and anti-sense gene expression, and altered cell cycle progression compared to HTLV-1.wt immortalized cells (CD4+ T-cells). These results suggest that the HTLV-1 enhancer element alone does not determine persistence or disease development but plays a pivotal role in regulating viral gene expression.