HTLV-1 Rex is required for viral spread and persistence in vivo but is dispensable for cellular immortalization in vitro

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is associated with leukemia/lymphoma and neurologic disorders. Although the viral transcriptional activator Tax is the critical viral oncoprotein, Rex, which regulates the expression of the viral structural and enzymatic genes, is essential for efficient viral replication. Herein, we investigate the contribution of Rex in HTLV-1 immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. A Rex-deficient HTLV-1 (HTLVRex-) was constructed and characterized for viral gene expression, protein production, and immortalization capacity. Cells transiently transfected with the HTLVRex- proviral clone produced low detectable levels of p19 Gag. 729HTLVRex- stable transfectants produced functional Tax, but undetectable levels of Rex or p19 Gag. Coculture of irradiated 729HTLVRex- cells with peripheral blood mononuclear cells (PBMCs) resulted in sustained interleukin-2 (IL-2)-dependent growth of primary T lymphocytes. These cells carried the HTLVRex- genome and expressed tax/rex mRNA but produced no detectable Rex or p19 Gag. Rabbits inoculated with irradiated 729HTLVRex- cells or 729HTLVRex- cells transiently transfected with a Rex cDNA expression plasmid failed to become persistently infected or mount a detectable antibody response to the viral gene products. Together, our results provide the first direct evidence that Rex and its function to modulate viral gene expression and virion production is not required for in vitro immortalization by HTLV-1. However, Rex is critical for efficient infection of cells and persistence in vivo.

Figure 1. Organization of the HTLV-1 genome and expanded Rex coding region

The complete proviral genome is shown schematically. LTRs are depicted with their U3, R, and U5 regions. The location of the gag, pro, pol, env, tax, and rex genes and their corresponding reading frames are indicated along with ORF I and ORF II. Numbers below the genome denote kilobases. The genome containing the 2 rex coding exons has been expanded, and the location of Rex based on the nucleotide sequence of the HTLV-1 proviral clone Ach is presented. The nucleotide sequence around the Rex start site (ATG) for wtHTLV-1 and mutant HTLVRex⁻ proviral clone are indicated. Vertical arrows denote the location of Rex protein start sites (ATG) and the SphI diagnostic restriction enzyme site (CGATGC). Asterisk denotes stop codon (TAG) in HTLVRex⁻. Location and orientation of oligonucleotide primers used in PCR and RT-PCR are indicated by horizontal arrows. Numbers below the Rex coding region denote amino acid number.

Figure 2. p19 Gag expression in 293T cells

2 × 10⁵ 293T cells were transfected with 5 µg of wtHTLV-1, HTLVRex⁻, or expression vector control DNA . At 72 hours after transfection, p19 Gag production was measured in the supernatant by ELISA. The values, which represent p19 Gag levels for 3 independent experiments, are normalized for transfection efficiency. Error bars indicate standard deviations. The data indicate detectable Gag production in the absence of Rex.

Figure 3. 729HTLVRex⁻ stable cell line contains the Rex mutation and does not produce detectable Rex

(A) HTLV-1 genome fragment containing the Rex start codon was amplified by PCR from genomic DNA of 729wtHTLV-1 and 729HTLVRex⁻ cells. PCR-amplified product was incubated in the presence (+) or absence (−) of SphI, separated on 2% agarose gel, and visualized by ethidium bromide staining. (B) Rex-1 immunoprecipitation. 729, 729wtHTLV-1, and 729HTLVRex⁻ cells were metabolically labeled with [³⁵S] methionine-cysteine. Cell lysates were immunoprecipitated with polyclonal antibody against the HTLV-1 Rex in the presence of protein A–sepharose. The sizes (in daltons, indicated on the left) were determined by comparison to protein markers.

Figure 4. Growth curve of HTLV T-lymphocyte immortalization assay

Human PBMCs were isolated by Ficoll/Paque and cocultivated with irradiated (10 000 rad) 729 stable cell lines (729wtHTLV-1, 729HTLVRex⁻) or 729 uninfected control cells as indicated. PBMCs (2 × 10⁶) were cocultured with irradiated donor cells (1 × 10⁶) in 24-well plates. Cells were fed once per week with RPMI 1640 supplemented to contain 20% FCS. Cell viability was determined by trypan blue exclusion staining at 0, 1, 2, 3, 4, 6, and 8 weeks after cocultivation. After 4 weeks, 10 U/mL IL-2 was provided in the culture medium. The mean and standard deviation of each time point were determined from 3 independent samples.

Figure 5. T lymphocytes immortalized by HTLVRex⁻ contain the expected Rex⁻ mutation and express tax/rex mRNA

(A) HTLV-1 genome fragment containing the Rex start codon was amplified by PCR from high molecular weight DNA of wtHTLV-1 and HTLVRex⁻ immortalized cells. PCR-amplified product was incubated in the presence or absence of SphI, separated on 2% agarose gel, and visualized by ethidium bromide staining. (B) Detection of tax/rex mRNA in the wtHTLV-1 and HTLVRex⁻ immortalized PBMCs. Total RNA was prepared from fresh PBMCs, wtHTLV-1, and HTLVRex⁻ immortalized PBMCs. Approximately 0.3 µg RNA was subjected to a coupled 40-cycle RT-PCR in the presence (+) or absence (−) of reverse transcriptase (RT). Following RT-PCR, 25-cycle nested PCR was performed. PCR product was separated on 2% agarose gel and visualized by ethidium bromide staining.

Figure 6. Assessment of HTLV-1 infection in rabbits

Rabbits were inoculated with 1 × 10⁷ irradiated 729wtHTLV-1, 729HTLVRex⁻, or 729HTLVRex⁻ + Rex (Table 1). At weeks 1, 2, 3, 4, 6, 8 after inoculation, rabbit PBMCs and sera were isolated from blood. (A) p19 Gag expression in the supernatant of ex vivo–cultured rabbit PBMCs. PBMCs were cultured ex vivo for 2 weeks, and p19 Gag in the culture supernatant was detected by ELISA. Results are the average of 2 rabbits for each cell line, indicating that only PBMCs from 729wtHTLV-1–inoculated rabbits produce detectable p19 Gag. Error bars indicate standard deviations. (B) HTLV-1–specific serologic response. Sera from inoculated rabbits were tested for reactivity to specific HTLV-1 proteins by Western blot. A representative rabbit from each group as indicated is shown with reactive viral proteins labeled on the left. Results indicate that only 729wtHTLV-1–inoculated rabbits are seropositive. (C) Detection of HTLV-1–specific sequences in rabbits. Genomic DNA was isolated from rabbit PBMCs and subjected to PCR using HTLV-1–specific oligonucleotide primers. A representative result from one rabbit in each group is shown. Viral DNA integration was only detected in wtHTLV-1–inoculated rabbits.