Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13(II) is required for viral infectivity in vivo

Abstract

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia, encodes unique regulatory and accessory proteins in the pX region of the provirus, including the open reading frame II product p13(II). p13(II) localizes to mitochondria, binds farnesyl pyrophosphate synthetase, an enzyme involved in posttranslational farnesylation of Ras, and alters Ras-dependent cell signaling and control of apoptosis. The role of p13(II) in virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13(II) in HTLV-1 infection. We compared the infectivity of a human B-cell line that harbors an infectious molecular clone of HTLV-1 with a selective mutation that prevents the translation of p13(II) (729.ACH.p13) to the infectivity of a wild-type HTLV-1-expressing cell line (729.ACH). 729.ACH and 729.ACH.p13 producer lines had comparable infectivities for cultured rabbit peripheral blood mononuclear cells (PBMC), and the fidelity of the start codon mutation in ACH.p13 was maintained after PBMC passage. In contrast, zero of six rabbits inoculated with 729.ACH.p13 cells failed to establish viral infection, whereas six of six rabbits inoculated with wild-type HTLV-1-expressing cells (729.ACH) were infected as measured by antibody responses, proviral load, and HTLV-1 p19 matrix antigen production from ex vivo-cultured PBMC. Our data are the first to indicate that the HTLV-1 mitochondrion-localizing protein p13(II) has an essential biological role during the early phase of virus infection in vivo.

FIG. 1.

Schematic illustrations of the HTLV-1 proviral genome (A) and the p13^II transcript (B), highlighting the mutation (CATG → AGAT) created for the mutant molecular clone (i.e., ACH → ACH.p13) that selectively ablates p13^II expression. The mutation also generates a unique BglII restriction site. LTR, long terminal repeat.

FIG. 2.

(A) Representative agarose gel image showing PCR products with or without the diagnostic BglII restriction enzyme digestion for detection of HTLV-1 ORF II sequences. 729(−), uninfected, HTLV-1-negative cell line. The presence of a unique BglII site within ORF II of ACH.p13 results in a cleavage of PCR products. (B) Comparable levels of production of HTLV-1 p19 matrix antigen by the cell lines as measured by p19 ELISA. Cells (5 × 10⁵) were seeded in 1 ml RPMI medium, cultured for 72 h, and analyzed for cell-free HTLV-1 p19 Gag antigen production. Each bar represents the average amount of p19 gag (±standard deviation), done in triplicate. The values were not statistically different (P > 0.05; Student's t test). The HTLV-1 proviral copy number for each cell line was determined by HTLV-1 tax-specific real-time PCR as described in Materials and Methods. (C) Western immunoblot image showing comparable levels of expression of HTLV-1 viral proteins produced by the inoculum cell lines. 729.ACH and 729.ACH.p13 were compared for their production of the HTLV-1 transactivator protein p40^Tax and envelope glycoprotein gp46. Equal loadings of proteins were verified using anti-β-actin antibody.

FIG. 3.

Decay of p19 Gag antigen production after γ irradiation of 729.ACH and 729.ACH.p13 cell lines. Cell culture supernatants were taken 24 h after complete medium changes. Values are the means from triplicate samples ± standard deviations. OD, optical density; 729(−), uninfected, HTLV-1-negative cell line.

FIG. 4.

(A) Antibody response against HTLV-1 from each rabbit was measured by anti-HTLV-1 ELISA, using both HTLV-1 whole-virus lysates and envelope protein as antigens. Each bar represents an average absorbance value for six samples from each group (729.ACH or 729.ACH.p13) ± the standard deviation. (B) Anti-HTLV-1 Western immunoblot. Rabbit sera were tested for antibody response to specific HTLV-1 viral antigens. Representative results from selected animals are shown. Rabbit H5 was inoculated with wild-type 729.ACH cells, whereas H12 and H13 were inoculated with 729.ACH.p13 cells. rgp46, HTLV-1-specific recombinant envelope surface protein; p53, Gag precursor; p24, capsid; p19, matrix; GD21, recombinant envelope transmembrane protein; 729(−), uninfected, HTLV-1-negative cells; *, serum loading control bands indicating comparable concentrations of serum immunoglobulin levels among the samples; **, nonspecific seroreactivity against human cell (i.e., the inoculum) antigen; ID, identification; p.i., postinoculation.

FIG. 5.

HTLV-1 p19 matrix antigen production in ex vivo-cultured rabbit PBMC from each sampling time point. PBMC from each infected rabbit were cultured for 2 weeks at each sampling point, and production of HTLV-1 p19 matrix antigen in culture supernatant was measured by ELISA. Bars represent the mean absorbance values from six rabbits in either the 729.ACH- or the 729.ACH.p13- inoculated group ± standard deviations for preinoculation (week 0) and weeks 2, 4, and 8 postinoculation.

FIG. 6.

(A) HTLV-1 proviral loads in infected rabbit PBMC. Viral loads in the infected rabbit PBMC were determined and compared using HTLV-1 tax-specific quantitative real-time PCR. Average viral copy numbers of six rabbits for each inoculum from preinoculation (week 0) and 2- and 8-week postinoculation samplings are shown. *, below detection sensitivity (81 proviral copies per 10⁶ PBMC). Horizontal bars indicate average copy numbers (n = 6). (B) PCR amplification (445-bp products) of the HTLV-1 provirus from 729.ACH-inoculated rabbit PBMC DNA (H3 to H8) compared to levels of amplification of the virus with the designated mutation site from the rabbit PBMC DNA (H9 to H14) at 8 weeks postinoculation. Rabbits H3 through H8 and H9 through H14 were inoculated with 729.ACH and 729.ACH.p13 cells, respectively. Rabbit H2 was inoculated with the mock control cell line. The plasmids (ACH and ACH.p13) were used as positive controls. MM, PCR master mix.

FIG. 7.

In vitro coculture experiment. Freshly isolated rabbit PBMC were cocultured with γ-irradiated HTLV-1 producer cells (729ACH or 729.ACH.p13) for in vitro infectivity assay. A graph shows p19 matrix antigen production over 6 weeks of coculture. Average absorbance values ± standard deviations of quadruplicate samples are shown. P values were >0.05 for each time point (Student's t test). OD, optical density; 729(−), uninfected, HTLV-1-negative cells.