The primary mode of infection by human T-cell leukemia virus type 1 (HTLV-1) is cell-to-cell transmission during contact between infected cells and target cells. Cell-free HTLV-1 infections are known to be less efficient than infections with other retroviruses, and transmission of free HTLV-1 is considered not to occur in vivo. However, it has been demonstrated that cell-free HTLV-1 virions can infect primary lymphocytes and dendritic cells in vitro, and that virions embedded in biofilms on cell membranes can contribute to transmission. The establishment of an efficient cell-free HTLV-1 infection model would be a useful tool for analyzing the replication process of HTLV-1 and the clonal expansion of infected cells. We first succeeded in obtaining supernatants with high-titer cell-free HTLV-1 using a highly efficient virus-producing cell line. The HTLV-1 virions retained the structural characteristics of retroviruses. Using this cell-free infection model, we confirmed that a variety of cell lines and primary cultured cells can be infected with HTLV-1 and demonstrated that the provirus was randomly integrated into all chromosomes in the target cells. The provirus-integrated cell lines were HTLV-1-productive. Furthermore, we demonstrated for the first time that cell-free HTLV-1 is infectious in vivo using a humanized mouse model. These results indicate that this cell-free infection model recapitulates the HTLV-1 life cycle, including entry, reverse transcription, integration into the host genome, viral replication, and secondary infection. The new cell-free HTLV-1 infection model is promising as a practical resource for studying HTLV-1 infection.IMPORTANCECo-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection. Co-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection.
