Kinetic analysis of human T-cell leukemia virus type 1 gene expression in cell culture and infected animals

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) infection causes adult T-cell leukemia and is associated with a variety of lymphocyte-mediated disorders. It has been hypothesized that a highly regulated pattern of HTLV-1 gene expression is critical for virus survival and disease pathogenesis. In this study, real-time reverse transcriptase PCR was used to determine the kinetics of viral gene expression in cells transiently transfected with an HTLV-1 proviral plasmid, in newly infected human peripheral blood mononuclear cells (PBMCs), and in PBMCs from newly infected rabbits. The HTLV-1 gene expression profiles in transiently transfected and infected cells were similar; over time, all transcripts increased and then maintained stable levels. gag/pol, tax/rex, and env mRNA were detected first and at the highest levels, whereas the expression levels of the accessory genes, including the antisense Hbz, were significantly lower than the tax/rex levels (ranging from 1 to 4 logs depending on the specific mRNA). In infected rabbits, tax/rex and gag/pol mRNA levels peaked early after inoculation and progressively decreased, which correlated inversely with the proviral load and host antibody response against viral proteins. Interestingly, Hbz mRNA was detectable at 1 week postinfection and increased and stabilized. The expression levels of all other HTLV-1 genes in infected rabbit PBMCs were at or below our limit of detection. This analysis provides insight into viral gene expression under various in vitro and in vivo experimental conditions. Our in vivo data indicate that in infected rabbits, Hbz mRNA expression over time directly correlates with the proviral load, which provides the first evidence linking Hbz expression to proviral load and the survival of the virus-infected cell in the host.

FIG. 1.

Provirus genome of HTLV-1 and its unspliced, singly spliced, and doubly spliced mRNA. At least eight positive-sense transcripts and one negative-sense transcript are expressed by HTLV-1. The genomic unspliced mRNA encodes the Gag, Pol, and Pro proteins. Four singly spliced mRNA species are the result of splicing of exon 1 (nucleotides 1 to 119) to major splice acceptors at positions 4641 (Env), 6383 or 6478 (p12), 6950 (p21rex), and 6875 (p13). The three doubly spliced mRNA include exon 1, exon 2 (4641 to 4831), and a third exon that starts at position 6950 (Tax/Rex), 6478 (p30), or 6383 (p27/p12). The Hbz singly spliced major antisense transcript initiates at multiple sites in the 3′ LTR and utilizes a splice donor site at position 365 and a splice acceptor site at nucleotide position 1765 (Hbz unspliced and minor spliced transcripts not shown). Nucleotide numbering starts at the beginning of the R region for the positive-sense transcripts and the last nucleotide of U5 in the 3′ LTR for the antisense transcript. Black lines designate exons, and dotted lines designate introns. Major utilized splice donor (open triangles) and splice acceptor (closed triangles) sites are indicated above the unspliced mRNA. An, poly(A) adenylation; spHbz, spliced Hbz.

FIG. 2.

Time course of HTLV-1 protein expression following transient transfection. 293T cells (8 × 10⁵) were transfected with 8 μg HTLV-1 ACHneo plasmid by using Lipofectamine in 100-mm plates. Culture supernatants and cell lysates were harvested at 12, 20, 28, 36, 44, and 60 h posttransfection. (A) Detection of HTLV-1 p24, Rex, and Tax protein expression in transfected-cell lysates by Western blot analysis. β-Actin levels were assessed as a loading control. +, present; −, absent; M, marker. (B) p19 Gag ELISA was used to quantify HTLV-1 viral particle production in culture supernatant. p19 was first detected in the supernatant at 12 h posttransfection. Error bars indicate standard deviations of the results of two independent transfections performed in triplicate.

FIG. 3.

HTLV-1 T-lymphocyte immortalization assay. Freshly isolated human PBMCs (2 × 10⁶) were cultured with 10⁶ γ-irradiated 729HTLV-1 producer cells in 24-well plates. (A) Representative growth curve is presented to show cell viability as determined by trypan blue exclusion at weekly intervals. Numbers shown at each time point are the average of the results from three random wells, with error bars denoting standard deviations. (B) p19 Gag ELISA was used to quantify HTLV-1 virion production by detecting Gag protein in the culture supernatants. Numbers shown at each time point are the averages of the results from three random wells, with error bars denoting standard deviations. First time point begins at 3 weeks when there is no remaining signal in the supernatant from irradiated producer cells (49). (C) Real-time RT-PCR was performed on cells from triplicate wells at weekly intervals (1 to 8) and at a final 14-week time point to quantify the HTLV-1 transcript levels throughout the immortalization process. The data are presented graphically, with error bars denoting standard deviations, and with the average numbers displayed below. Human GAPDH mRNA was used as the internal control, and numbers presented are normalized to 1 × 10⁶ copies of human GAPDH mRNA.

FIG. 4.

Assessment of HTLV-1 infection in rabbits. Twelve-week-old New Zealand White rabbits were inoculated with 1 × 10⁷ γ-irradiated 729HTLV-1 producer cells (nine total rabbits) or uninfected 729 cells as a control (two total rabbits). Following inoculation, 15 ml of blood was drawn from each rabbit at week 0, 1, 2, 4, 6, and 8 for collection of sera and rabbit PBMCs. (A) Sera from inoculated rabbits were tested for reactivity to specific HTLV-1 proteins by Western blotting. Results for a representative rabbit from each group, as indicated, are shown with reactive viral proteins labeled on the right. rgp46, HTLV-1-specific recombinant envelope surface protein; p53, Gag precursor; p24, capsid; p19, matrix; GD21, recombinant envelope transmembrane. Results labeled “Serum control” indicate comparable concentrations of serum Ig levels among the samples. (B) Genomic DNA was isolated from rabbit PBMCs and subjected to TaqMan real-time PCR using HTLV-1-specific primers #19 and #20 and probe TMP-3; the standard curve was generated by serial 10-fold dilutions of plasmid DNA (27). The proviral load is plotted against weeks postinfection, with the actual copy number per cell displayed below. The copy number per cell value for a sample was generated based on the estimation that 1 μg of PBMC DNA is equivalent to 134,600 cells. R1 to R11, rabbits 1 to 11.

FIG. 5.

Kinetics and profile of HTLV-1 mRNA expression in infected rabbits. RNA extracted from rabbit PBMCs (harvested from rabbits at week 0, 1, 2, 4, 6, and 8 postinoculation as described in Fig. 4 legend) were subject to real-time RT-PCR to quantitate tax/rex mRNA expression (A), gag/pol and full-length genomic mRNA expression (B), and Hbz antisense mRNA expression (C). Numbers shown are values normalized to 1 million copies of rabbit GAPDH mRNA. R1 to R11, rabbits 1 to 11.

FIG. 6.

Relationship between HTLV-1 proviral load and tax/rex or Hbz mRNA levels in infected rabbits. We used log transformation of both proviral load (data used are those in Fig. 4) and tax/rex or Hbz mRNA (data used are those in Fig. 5) to stabilize the variance. Linear mixed modeling was employed to take account of correlated multiple observations in a time series from the same rabbit. (A) The data show a negative correlation between log tax/rex mRNA expression and log proviral load in HTLV-1-infected rabbits (P = 0.0003). (B) The data show a direct correlation between log Hbz mRNA and log proviral load in HTLV-1-infected rabbits (P = 0.0001). Each weekly time point is designated by a distinct symbol, as indicated, and the result for each individual infected rabbit per weekly time point is denoted (1 to 9) inside the symbol.