As a result of alternative splicing, HTLV-2 p28 has the potential to be expressed from two distinct singly-spliced mRNAs (Fig. 1). Both mRNAs also have the potential to produce the amino terminal truncated p22/p20 Rex proteins 2843. It is important to note that the p28 ORF has complete overlap with Tax exon 3 and partial overlap with Rex exon 3 (Fig. 1). Using an over-expression system approach, previous studies revealed that p28 is at least in part functionally homologous to HTLV-1 p30 and has the capacity to specifically retain tax/rex mRNA in the nucleus, thus decreasing Tax and Rex protein and viral replication via a posttranscriptional mechanism 30. However, the specific role of p28 in the context of a proviral clone, and ultimately on virus biology, has not been investigated. In order to determine the potential role of p28 in HTLV-2-mediated cellular immortalization in cell culture and viral persistence in inoculated rabbits, a p28-deficient proviral clone (HTLV-2Δp28) was generated from the HTLV-2 molecular clone pH6neo. To construct HTLV-2Δp28, a single nucleotide was altered by site directed mutagenesis, which introduced a stop codon at amino acid 7 of the p28 ORF and had no affect on the overlapping Tax and Rex amino acid sequence. We initially determined whether knocking out p28 altered Tax and/or Rex activities. Co-transfection of wild-type HTLV-2 or HTLV-2Δp28, as a source of Tax, and the LTR-2-Luc reporter revealed that HTLV-2Δp28 had a consistently lower, but not significantly different LTR-directed gene expression (Fig. 2A). Moreover, cells transfected with HTLV-2Δp28 produced levels of p19 Gag in the culture supernatant similar to wild-type HTLV-2, indicating no significant repression of Rex function (Fig. 2B). Based on the reported functional activity of over-expressed p28, we were surprised that deletion of p28 did not translate into an increase in Tax activity or p19 Gag expression. Although p28 mRNA is easily detectable following transient transfection with proviral clones (Fig. 2C), we have been unable to detect p28 protein by Western blot 30 (Fig. 2C and data not shown). We then determined the effect of exogenously over-expressed p28- and Δp28-AU1 tagged proteins on Tax-mediated transcription. Our results confirmed previous reports that over-expressed p28 from a CMV-cDNA expression vector significantly repressed Tax activity in a dose-dependent manner (Fig. 3A). Importantly, the Δp28 cDNA expression vector failed to repress Tax activity (Fig. 3A). Western blot analysis confirmed the expression of p28 and that the Δp28 amino terminal truncation mutation resulted in a complete loss of p28 protein expression (Fig. 3B). Therefore, our results are consistent with the conclusion that either p28 protein is not expressed from the proviral clone following transient transfection (48 hours) or that the levels of p28 expressed from the proviral clone are below the threshold concentration required for detection by Western blot and necessary for repression of Tax or Rex activity.

To determine the capacity of HTLV-2Δp28 to synthesize viral proteins, direct viral replication, and induce cellular immortalization, stable 729 cell transfectants expressing wild-type and p28-deleted HTLV-2 proviral clones were generated and characterized. Four independent stable HTLV-2Δp28 transfectants were isolated and found to contain complete copies of the provirus; the presence of the expected Δp28 mutation was confirmed by sequencing (data not shown). We quantified the concentration of p19 Gag produced in the culture supernatant of the four cell clones by ELISA. Our results showed p19 Gag expression ranging from 250–750 pg/ml (Fig. 4A). The variable p19 Gag expression from independent stable cell clones was attributed to chromosomal location of proviral sequences and overall proviral copy number, consistent with previous analyses 4445. We did not observe a pattern of increased viral gene expression in the absence of p28. For additional studies, we selected 729.HTLV-2Δp28Clone 3, a stable producer line with p19 Gag production similar to that of our well-characterized wild-type HTLV-2-producer cell line 729pH6neo (729.HTLV-2). Further characterization revealed that, as with transient transfection, p28 mRNA was detected at similar levels (approximately 10³ copies per 10⁶ copies of cellular gapdh mRNA) in 729.HTLV-2 and 729.HTLV-2Δp28Clone 3 (Fig. 4B), but Western blot analyses failed to detect p28 protein in 729.HTLV-2 or 729.HTLV-2Δp28 (data not shown). A similar level of Tax-2 expression in 729.HTLV-2 and 729.HTLV-2Δp28 relative to the β-actin loading control was detected by Western blot and, as expected, Tax-2 was not detected in the 729 negative control cells (Fig. 4B). Therefore, as with transient transfection, the repressive effect of p28 expressed from a stably integrated provirus on Tax-mediated transcription was not detectable.

We assessed the ability of the HTLV-2Δp28 to induce proliferation and immortalize human PBMCs in co-culture assays. Freshly isolated human PBMCs co-cultured with lethally irradiated 729.HTLV-2 or 729.HTLV-2Δp28 in the presence of 10 U/ml of human IL-2 showed very similar progressive growth patterns consistent with the HTLV-2 immortalization process, whereas control cells died within the first few weeks (Fig. 5A). Immortalized PBMCs expressed similar levels of p19 Gag and harbored the expected HTLV-2 sequences indicating that viral transmission was responsible for the immortalization of PBMCs (data not shown). In an effort to obtain a more quantitative measure of the ability of these viruses to infect and immortalize PBMCs, a fixed number of PBMCs were co-cultured with virus-producing cells in a 96-well plate assay 45. Since this assay is very stringent as a result of diluting the cultures 1:3 weekly, slowly growing or non-dividing cells are eliminated very quickly and the percentage of surviving wells is an accurate measure of the immortalization efficiency of viruses. A Kaplan-Meier plot of HTLV-2-induced T-cell proliferation or survival indicated that the percentage of wells containing proliferating lymphocytes was similar between HTLV-2 and two independently isolated HTLV-2Δp28 clones (Fig. 5B). Taken together, our results are consistent with the conclusion that p28 is not required for efficient infectivity or HTLV-2-mediated immortalization of primary human T-lymphocytes in culture.

To evaluate the role of p28 in vivo, we compared the abilities of 729, 729.HTLV-2, or 729.HTLV-2Δp28 cell lines to transmit virus to rabbits, which is an established model to investigate HTLV infection and persistence 46. Rabbits were inoculated with lethally irradiated cell lines (cell inocula were equilibrated based on their p19 Gag production) and on weeks 0, 1, 2, 4, 6, 8, and 11, whole blood was collected and processed for isolation of plasma and PBMCs. Antibody response to viral antigens was detectable by Western blot in all rabbits inoculated with cells expressing either wild type HTLV-2 or HTLV-2Δp28, and the antibody titers in the majority of the rabbits increased over the time course of the study (data not shown). Moreover, quantitative comparison of antibody responses between each rabbit was performed using an HTLV-specific ELISA (Fig. 6). Statistical analysis of titers at six, eight, and eleven weeks post-inoculation revealed a significantly lower antibody response to HTLV-2 antigens in the 729.HTLV-2Δp28-inoculated rabbits as compared to the wild-type HTLV-2 control group. Consistent with our antibody data, HTLV-2 proviral DNA sequences were detected in all wild type HTLV-2 and five of six HTLV-2Δp28-infected rabbits at two weeks post inoculation (Table 1). However, over time, HTLV-2Δp28 failed to persist and quantitative real-time Taqman PCR revealed that at eleven weeks post inoculation, proviral loads in rabbits infected with HTLV-2Δp28 were below the level of detection. Taken together, our results indicated that p28, while dispensable for HTLV-2 infection, attenuated virus replication as measured by antibody response to viral antigens and proviral loads. This attenuation was apparent within two weeks post inoculation, suggesting that p28 is required early for efficient replication and survival in the host.

293T cells and 729 B cell lines were maintained in Dulbecco's modified Eagle and Iscove medium, respectively, supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 ug/mL). Human and rabbit peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Hypaque (Amersham, Piscataway, NJ) and Percoll^® (Amersham, Piscataway, NJ), respectively, and cultured in RPMI 1640 medium supplemented with 20% FBS, glutamine and antibiotics as above, plus 10 U/mL of recombinant interleukin-2 (IL-2; Roche Applied Biosciences, Indianapolis, IN).

The p28 cDNA expression vector (CMV-p28-AU1) and the wild type (wt) HTLV-2 infectious proviral clone (pH6neo) were described previously 3048. Using PCR mutagenesis and CMV-p28-AU1 as a template, a single nucleotide mutation (C to A) was introduced in the p28 reading frame. This change (nt 7333 of the pH6neo proviral sequence) resulted in a stop codon in the seventh amino acid (aa) of p28, designated Δp28. This specific mutation was designed to not alter the aa sequence of either Tax or Rex, both of which share overlapping reading frames with p28. The Δp28 mutation expressed in the context of the proviral clone pH6neo, was designated HTLV-2Δp28. The mutation in all mutant plasmids was confirmed by DNA sequencing. The Tax reporter plasmid, LTR-2-Luc, and the transfection efficiency control plasmid, TK-Renilla, were described previously 3031.

293T cells (2 × 10⁵) were transfected using Lipofectamine^® (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. For p28 protein detection, cells were transfected with 1 μg of cDNA expression plasmids and 10 ng of TK-Renilla. Cell lysates were prepared at 48 h post transfection and normalized for transfection efficiency prior to Western blot analysis. To assess the repressive effects of p28 or Δp28 by Tax reporter assays, cells were transfected with 1 μg wtHTLV-2 in the presence or absence of variable concentrations (0.2–0.4 μg) of p28 or Δp28 cDNA expression vector and 0.1 μg of LTR-2-Luc, and 10 ng of TK-Renilla or 1 μg HTLV-2Δp28 and 0.1 μg of LTR-2-Luc, and 10 ng of TK-Renilla. Cell lysates were harvested at 48 h post transfection and dual luciferase activity was measured. The data represent average luciferase activity values after normalization for transfection efficiency for three independent experiments. To generate the 729HTLV-2Δp28 stable transfectant, the proviral plasmid clone containing neo^r gene was introduced into cells by nucleofection using the Nucleofector kit V (Amaxa Biosystems, Gaithersburg, MD). Stable transfectants containing the desired proviral clone were isolated following incubation in 24-well culture dishes in medium containing 1 mg/ml Geneticin (Gibco, Carlsbad, CA). Following a 4–5 weeks selection period, viable cells were expanded and maintained in culture for further analysis. The well-characterized wtHTLV-2 729 producer cell line (729pH6neo) used in this study was described previously 4649.

To detect p28, 50 μg of total cell lysates from transfected cells was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ). Rabbit polyclonal antibodies against p28 or a monoclonal antibody to AU1 (Covance Research Products, Denver, PA,) was used for p28 detection. Rabbit polyclonal antibody to β-actin (Novus Biological, Littleton, CO) was used as a loading control. Proteins were visualized using the ECL western blotting analysis system (Santa Cruz Biotechnology, Santa Cruz, CA).

DNA was isolated from 729 producer cells and rabbit peripheral blood mononuclear cells (PBMCs) using the PURGENE DNA purification system (Gentra, Minneapolis, MN). Rabbit DNA (1 μg) was subjected to a standard 40-cycle PCR amplification for detection of integrated provirus and the product was visualized on a 2% agarose gel stained with ethidium bromide. The primer pair used in the PCR, based on the pH6neo sequence, was TRE-PH-S (5'-⁴¹GAG TCA TCG ACC CAA AAG G⁵⁹-3') and TRE-PH-AS (5'-²⁹⁸TGC GCT TTT ATA GAC TCG GC²⁷⁹-3'), which amplified a 257 bp product in the HTLV-2 LTR. Taqman real-time PCR (Applied Biosystems, Foster City, CA) using 500 ng of rabbit DNA and 40 cycle amplification was performed in a 25 ul reaction to quantify the proviral copy number per cell in infected rabbit PBMCs using primers and probes directed towards Gag sequences 43. The reaction contained 100 ng (25 ng/μL) of each primer and the probe at a concentration of 100 pmol/μL. A standard curve was generated for each run using duplicate samples of log₁₀ dilutions of a plasmid containing the Gag sequences. The copy number for each sample was determined from the standard curve, and the copy number per cell for each sample calculated based on the estimate that 1 μg PBMC DNA is equal to 67,300 cells.

Short term microtiter proliferation assays were performed as detailed previously with modifications 4550. Briefly, freshly isolated human PBMCs were pre-stimulated with 2 μg/ml PHA and 10 U/ml IL-2 (Roche, Indianapolis, IN) for three days. 729 producer cells (2 × 10³) were irradiated (100 Gy) and co-cultured with 10⁴ pre-stimulated PBMCs in the presence of IL-2 in 96-well round bottom plates. Wells were enumerated for growth and split 1:3 at weekly intervals. Cell proliferation was confirmed by MTS assay using CellTiter 96^® Aqueous One Solution Reagent as recommended by the manufacturer (Promega, Madison, WI). For the long-term immortalization assays, 10⁶ irradiated producer cells were co-cultivated with 2 × 10⁶ freshly isolated PBMCs with 10 U/ml IL-2 in 24-well culture plates 41. HTLV expression was confirmed by detection of p19 Gag protein in the culture supernatant measured at weekly intervals using a commercially available ELISA (Zeptometrix, Buffalo, NY). Viable cells were counted weekly by trypan blue exclusion. Cells inoculated with HTLV-2 that continued to produce p19 Gag antigen and proliferate 12 weeks post co-culture in the presence of exogenous interleukin-2 (IL-2) were identified as HTLV immortalized. For each assay, at least three independent experiments were performed using PBMCs from distinct healthy donors.

Twelve week-old specific pathogen-free New Zealand White rabbits (Harlan, Indianapolis, IN) were inoculated with approximately 1 × 10⁷ gamma-irradiated (100 Gy) 729 viral producer cells (6 rabbits per group) or 729 uninfected control cells (2 rabbits) via the lateral ear vein. The virus-containing inocula were equilibrated based on HTLV-2 p19 Gag production (ELISA). At weeks 0, 1, 2, 4, 6, 8, and 11 after inoculation, 10 ml of blood was drawn from the central auricular artery from each animal and rabbit plasma and PBMCs were isolated. HTLV Western blot assay (HTLV Blot 2.4 Western Blot Assay; MP Diagnostics, Singapore) was used to examine serum reactivity to specific viral antigenic determinants. Serum showing reactivity to Gag (p24 or p19) and Env (gp21 or gp46) antigens was classified as positive for HTLV-2 seroreactivity. A commercial HTLV ELISA kit (Vironostika HTLV-I/II Microelisa System; bioMerieux, Durham, NC) was used to quantitate HTLV-2 serum antibody using plasma diluted 1:100 to obtain values within the linear range of the assay. Data is shown as absorbance values. DNA was isolated from rabbit PBMCs using the PURGENE DNA purification system (Gentra, Minneapolis, MN) and subjected to proviral load analysis by realtime PCR.