Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.

Fig 1. Tax and SBZ expression in STLV-1 naturally infected monkeys.

SBZ and tax expression level in various tissues and organs of STLV-1 naturally infected Japanese macaques (JM1-3) was measured by quantitative PCR. The relative expression level per provirus was presented.

Fig 2. HTLV-1 infection in multiple lineage hematopoietic cells.

Bone marrow cells from two STLV-1 infected JMs were cultured for 24 hours after removal of CD8⁺ T cells. (A, B) Tax expression in bone marrow cells of two STLV-1 infected JMs. Mononuclear cells from bone marrow were stained by various antibodies. Tax expression was shown in association with CD3, CD4, CD8, CD34 and CD33 expression.

Fig 3. The proportions of clones with the same integration sites.

The proportions of clones with the same integration sites are shown. The same integration sites are identified in 5 (red), 4 (yellow), 3 (light blue), and 2 (blue) different lineage cells. For HAM/TSP1, the proportion of clones with the same integration sites between PBMCs and neutrophils are shown in orange.

Fig 4. HTLV-1 infection in neutrophils and monocytes.

(A) Tax expression is detected by confocal immunofluorescence microscopy in MT4 and neutrophils from HAM/TSP patients using anti-Tax antibody and secondary Alexa Fluor 488 antibody. Myeloperoxidase expression is also detected in neutrophils using anti-MPO antibody and secondary Alexa Fluor 568 antibody. Jurkat was used as a negative control. MT4, HTLV-1 infected cell line; Jurkat cells, HTLV-1 negative human T-cell line. DAPI (blue) was used for nuclei staining. (B) JET WT35 cells were co-cultured with HPB-ATL-2 cells in the presence and absence of azidothymidine (AZT) or raltegravir (RAL), or HTLV-1 uninfected T cell line, CCRF-CEM. The number of tdTomato-positive cells is presented. (C) Dendritic cells were induced from monocytes from HAM/TSP patients, and then differentiated DCs were co-cultured with JET WT35. After 48 hours, newly infected JET WT35 was shown to be red. (D) Quantitative data of co-culture between differentiated DCs and JET WT35 cells. The count of tdTomato positive cells and the number of cells used for this experiment are shown.

Fig 5. Persistence of HTLV-1 infected clones in HAM/TSP patients at different time point.

HTLV-1 integration sites are determined using high throughput sequencing using genomic DNAs from neutrophils one year later in two HAM/TSP patients. The cell number of each clone is demonstrated by color. The longitudinal column represents the same integration site.