Amanda R. Panfil

HTLV-1 is an oncogenic human retrovirus and the etiologic agent of the highly aggressive ATL malignancy. Two viral genes, Tax and Hbz, are individually linked to oncogenic transformation and play an important role in the pathogenic process. Consequently, regulation of HTLV-1 gene expression is a central feature in the viral lifecycle and directly contributes to its pathogenic potential. Herein, we identified the cellular transcription factor YBX1 as a binding partner for HBZ. We found YBX1 activated transcription and enhanced Tax-mediated transcription from the viral 5′ LTR promoter. Interestingly, YBX1 also interacted with Tax. shRNA-mediated loss of YBX1 decreased transcript and protein abundance of both Tax and HBZ in HTLV-1-transformed T-cell lines, as well as Tax association with the 5′ LTR. Conversely, YBX1 transcriptional activation of the 5′ LTR promoter was increased in the absence of HBZ. YBX1 was found to be associated with both the 5′ and 3′ LTRs in HTLV-1-transformed and ATL-derived T-cell lines. Together, these data suggest that YBX1 positively influences transcription from both the 5′ and 3′ promoter elements. YBX1 is able to interact with Tax and help recruit Tax to the 5′ LTR. However, through interactions with HBZ, YBX1 transcriptional activation of the 5′ LTR is repressed.

HTLV‐I‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neuroinflammatory demyelinating condition of the spinal cord. We have previously shown that aberrant expression and activity of immune checkpoint (ICP) molecules such as PD‐1 and PD‐L1/PD‐L2, negatively associates with the cytolytic potential of T cells in individuals with HAM/TSP. Interestingly, ICPs can exist in a soluble cell‐free form and can be carried on extracellular vesicles (EVs) and exosomes (small EVs, <300 nm) while maintaining their immunomodulatory activity. Therefore, we investigated the role of soluble and exosomal ICPs in HTLV‐1 associated neuroinflammation. For the very first time, we demonstrate a unique elevated presence of several stimulatory (CD27, CD28, 4‐1BB) and inhibitory (BTLA, CTLA‐4, LAG‐3, PD‐1, PD‐L2) ICP receptors in HAM/TSP sera, and in purified exosomes from a HAM/TSP‐derived HTLV‐1‐producing (OSP2) cells. These ICPs were found to be co‐localized with the endosomal sorting complex required for transport (ESCRT) pathway proteins and exhibited functional binding with their respective ligands. Viral proteins and cytokines (primarily IFNγ) were found to be present in purified exosomes. IFNγ exposure enhanced the release of ICP molecules while antiretroviral drugs (Azidothymidine and Lopinavir) significantly inhibited this process. HTLV‐1 b‐Zip protein (HBZ) has been linked to factors that enhance EV release and concurrent knockdown here led to the reduced expression of ESCRT associated genes (e.g., Hrs , Vsp4, Alix, Tsg101 ) as well as abrogated the release of ICP molecules, suggesting HBZ involvement in this process. Moreso, exosomes from OSP2 cells adversely affected CD8 T‐cell functions by diminishing levels of cytokines and cytotoxic factors. Collectively, these findings highlight exosome‐mediated immunomodulation of T‐cell functions with HBZ and ESCRT pathways as an underlying mechanism in the context of HTLV‐1‐induced neuroinflammation.