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. 2006 Nov;80(21):10683-91.
doi: 10.1128/JVI.01009-06. Epub 2006 Aug 30.

De novo human T-cell leukemia virus type 1 infection of human lymphocytes in NOD-SCID, common gamma-chain knockout mice

Paola Miyazato  1 , Jun-ichirou Yasunaga, Yuko Taniguchi, Yoshio Koyanagi, Hiroaki Mitsuya, Masao Matsuoka
Affiliations

De novo human T-cell leukemia virus type 1 infection of human lymphocytes in NOD-SCID, common gamma-chain knockout mice

Paola Miyazato et al. J Virol. 2006 Nov.

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, a disease that is triggered after a long latency period. HTLV-1 is known to spread through cell-to-cell contact. In an attempt to study the events in early stages of HTLV-1 infection, we inoculated uninfected human peripheral blood mononuclear cells and the HTLV-1-producing cell line MT-2 into NOD-SCID, common gamma-chain knockout mice (human PBMC-NOG mice). HTLV-1 infection was confirmed with the detection of proviral DNA in recovered samples. Both CD4+ and CD8+ T cells were found to harbor the provirus, although the latter population harbored provirus to a lesser extent. Proviral loads increased with time, and inverse PCR analysis revealed the oligoclonal proliferation of infected cells. Although tax gene transcription was suppressed in human PBMC-NOG mice, it increased after in vitro culture. This is similar to the phenotype of HTLV-1-infected cells isolated from HTLV-1 carriers. Furthermore, the reverse transcriptase inhibitors azidothymidine and tenofovir blocked primary infection in human PBMC-NOG mice. However, when tenofovir was administered 1 week after infection, the proviral loads did not differ from those of untreated mice, indicating that after initial infection, clonal proliferation of infected cells was predominant over de novo infection of previously uninfected cells. In this study, we demonstrated that the human PBMC-NOG mouse model should be a useful tool in studying the early stages of primary HTLV-1 infection.

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Figures

FIG. 1.
FIG. 1.
Surface marker analysis of splenocytes in hu-PBMC-NOG mice. Splenocytes were isolated from hu-PBMC-NOG mice with or without HTLV-1 infection, and their surface markers were analyzed by flow cytometry. Splenocytes were recovered at 2 weeks p.i. The percentages of cells positive for various surface molecules are shown for MT-2-inoculated hu-PBMC-NOG mice (black bars) and uninfected controls (open bars). Values are means ± standard deviations from groups of three mice. *, P < 0.05 (Student's t test).
FIG. 2.
FIG. 2.
Polyclonal proliferation of HTLV-1-infected cells in the spleens of hu-PBMC-NOG mice (2W-1, 2W-2, 4W-1, and 4W-2). Genomic DNA was isolated from recovered splenocytes and analyzed by IL-PCR as described in Materials and Methods. IL-PCR was performed in triplicate for each DNA sample. Genomic DNA was recovered from splenocytes at 2 or 4 weeks after injection of MT-2 cells. D, DNA of donor PBMC before inoculation; W, water; P, positive control (DNA from PBMC of an HTLV-1 carrier). In addition, proviral load was quantified by real-time PCR as described in Materials and Methods and is shown as a relative percentage.
FIG. 3.
FIG. 3.
DNA methylation of HTLV-1 provirus. hu-PBMC-NOG mice were sacrificed 2 or 4 weeks after inoculation of MT-2 cells, and DNA methylation in the 5′ LTR, gag, and pol regions was studied by a COBRA assay. (+), positive control; U, intact fragment (unmethylated CpG); M, digested fragments (methylated CpG). Percentages of DNA methylation were calculated by densitography according to the following formula (with the variables as described above): [M/(U + M)] × 100.
FIG. 4.
FIG. 4.
Transcription of the tax gene increases after in vitro culture. Splenocytes of hu-PBMC-NOG mice inoculated with 104 MT-2 cells were recovered 2 weeks after infection. Transcription of the tax gene was quantified by semiquantitative PCR (A) or real-time PCR (B) at recovery and after 24 h of in vitro culture. Proviral loads for the same samples were also measured by real-time PCR. M, MT-1 cells; ID, identification number.
FIG. 5.
FIG. 5.
Cytotoxic effects of tenofovir (TFV) and AZT in vitro. Human PBMC were stimulated with PHA for 3 days. Cells were then cultured in medium alone or medium containing the specified concentration of the indicated drug for another three days, at a density of 105 cells/well, in a 96-well plate. Viability was assessed by MTT assay as described in Materials and Methods. The results show the means ± standard deviations of quadruplicate measurements made in one of three representative experiments.
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