Yukiko Inoue

Phosphatidylglycerol Participates in Syncytium Formation Induced by HTLV Type 1-Bearing Cells

We previously reported that 71-kDa heat shock cognate protein (HSC70) was expressed on the cell surface of human T cell lymphotropic virus type 1 (HTLV-1)-susceptible cells and that HSC70, beta-actin, and a lipid-like component on the target cell membrane participated in syncytium formation by HTLV-1. We have now identified this lipid-like component to be palmitoyl (16:0)-oleoyl (18:1)-phosphatidylglycerol (POPG), using preparative thin-layer chromatographic fractionation and tandem mass spectrometric analysis. In the syncytium formation assay, exogenously added PG inhibited cell-to-cell transmission of HTLV-1 in a dose-dependent manner. Other phospholipids showed less (PE) or no effect (PC, PS, PI, PA, lysoPC, lysoPE, and CL). Binding experiments showed that PG interacted with three synthetic peptides, gp46--111, gp46--197, and gp21--400, which correspond to regions Lys111--Asp138 and Asp197--Leu216 on the gp46 surface glycoprotein, and to region Cys400--Leu429 on the gp21 transmembrane glycoprotein, respectively, as well as with intact gp46 and gp21 proteins of HTLV-1. On the other hand, HSC70 and beta-actin interacted with gp46--197 and gp46, not with gp46--111. However, the eluate from an affinity column coupled with gp46--111 contained not only PG but also HSC70 and beta-actin, despite the lack of direct interaction between gp46--111 and these proteins. In the in vitro binding assay, HSC70 showed interaction with both PG and beta-actin, while there was no evidence of any interaction between PG and beta-actin. These results suggest that HSC70 molecules on target cell surface interact with both PG in lipid bilayers and intracellular beta-actin and that these three cellular components form a receptor complex that plays a critical role in syncytium formation induced by HTLV-1-bearing cells.

Involvement of molecular mimicry between human T-cell leukemia virus type 1 gp46 and osteoprotegerin in induction of hypercalcemia.

Human T‐cell leukemia virus type‐1 (HTLV‐1) causes adult T‐cell leukemia/lymphoma (ATL), frequently associated with hypercalcemia and bone destruction. A positive correlation between the appearance of an antibody recognizing the central region (Asp197 to Leu216) on Gp46, gp46‐197, and the severity of ATL has been demonstrated. In this study, five male Nihon Hakusyoku rabbits were immunized with a synthetic peptide corresponding to the gp46‐197 region to clarify its action and mechanism. Two of the rabbits showed piloerection, anorexia, and somnolence, and died soon after booster administration. The serum calcium level of the dead rabbits was significantly high, compared to those of surviving rabbits. Interestingly, amino acid sequences homologous with gp46‐197 were found in the carboxyl‐terminal half of osteoprotegerin (OPG), an osteoclast inhibitory factor. To confirm the effect of the gp46‐197 region on osteogenesis in vivo, the peptide was intraperitoneally administered to male Sprague‐Dawley rats. The administration of the gp46‐197 peptide resulted in a decrease of bone mineral density (BMD), a significant increase of serum calcium level, and inhibition of normal bone growth in both short‐ and long‐term experiments. In rats, femoral growth inhibition by the gp46‐197 peptide was restored by the coadministration of recombinant human OPG. Improvement by OPG in the adverse effect indicates that the central region of HTLV‐1 Gp46 acts as an antagonist for OPG and leads to hypercalcemia. (Cancer Sci 2009; 100: 490–496)

Novel biomarker of HTLV-1-associated disease: specific appearance of antibody recognizing the receptor-binding site on HTLV-1 envelope protein.

We previously showed that 71‐kDa heat shock cognate protein (HSC70) functions as a cellular receptor for gp46 protein via the gp46–197 region, corresponding to Asp197 to Leu216 of human T‐cell lymphotropic virus type 1 (HTLV‐1), leading to cell‐to‐cell transmission of HTLV‐1. We found that HSC70 protein was contained in goat serum and casein used as blocking agents in the usual ELISA method. Here, it was demonstrated that HSC70 contamination in the blocking agents causes a false‐negative result in the detection of anti‐gp46–197 antibody in serum samples from HTLV‐1‐infected individuals. By using ELISA without the blocking agents, we detected antibodies recognizing the HSC70‐binding site of gp46, and the anti‐gp46–197 antibody specifically appeared in sera from patients with HTLV‐1‐associated diseases. The frequency of serum anti‐gp46–197 antibody‐positive individuals was 98% and 100% among ATLL and HAM/TSP patients, respectively, but only 6% among asymptomatic HTLV‐1‐infected carriers (ACs). The antibody titer in ATLL and HAM/TSP patients was higher than that in ACs (P>0.002 for ATLL; P>0.0001 for HAM/ TSP). These findings suggest that appearance of the anti‐gp46–197 antibody is a predictive marker for the onset of HTLV‐1‐associated disease.

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing

Adult T‐cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T‐cell leukemia virus type 1 (HTLV‐1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti‐HTLV‐1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real‐time PCR and other tests in 600 HTLV‐1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV‐1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV‐1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV‐1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus‐positive samples, probably suggesting that antibody‐based screening tests should incorporate multiple HTLV‐1 antigens, such as Gag and Env antigens.

Antibody to the central region of human T‐lymphotropic virus type 1 gp46 is associated with the progression of adult T‐cell leukemia

Human T‐cell lymphotropic virus type 1 (HTLV‐1) is an etiologic agent of adult T‐cell leukemia/lymphoma (ATL). HTLV‐1 is spread by cell‐to‐cell transmission via the gp46–197 region, Asp197 to Leu216, on the envelope protein gp46. In the present study, we revealed a positive correlation between the appearance of an antibody recognizing the gp46–197 region (anti‐gp46–197 antibody) and the severity of ATL. The prevalence and titer of the anti‐gp46–197 antibody were found to be elevated along with the progression of ATL. In serial samples obtained from a single patient, the anti‐gp46–197 antibody was detected before treatment in acute phase, then diminished after allogeneic bone marrow transplantation, to which the patient had a complete response. However, the antibody appeared again before a relapse, along with an increase of the serum‐soluble interleukin‐2 receptor level and proviral load. The results from the other six patients also indicate that seroconversion of this antibody was synchronized with the deterioration of ATL. Taken together, the findings indicate that the anti‐gp46–197 antibody may be a novel beacon for gauging the efficacy of therapeutic approaches to ATL, and a survey of this antibody would be useful for identifying asymptomatic carriers infected with HTLV‐1 who are at high risk of developing ATL. (Cancer Sci 2007; 98: 240–245)

17beta-estradiol (E2) abrogated osteolysis induced by HTLV-1 Env protein in vivo

HTLV-1 is the causative retrovirus of ATL. Eighty percent of ATL patients develop hypercalcemia, a severe complication resulting from bone resorption. PTHrP is known as the major pathogenic factor to hypercalcemia associated malignancies. Recently, we observed hypercalcemia in rabbits with antibody against central region of HTLV-1 Env Gp46 (gp46-197), which shows a homology with osteoprotegerin (OPG), an inhibitor to the maturation of osteoclasts. To observe the effect of gp46197 synthetic peptide on osteogenesis, the peptide was intraperitoneally administered to mice in the presence or absence of co-administration of E2. Seven days later, serum E2 level and bone mineral density (BMD) were measured. Both of males and females exhibited a clear reduction in E2 and BMD by the gp46-197 peptide. Histomorphological analyses revealed that the peptide induced the loss of Ob.S/BS and increase in ES/BS, N. Oc/B.Pm, and Oc.S/BS. These results suggest that the structural mimicry of gp46-197 inhibits the OPG function and promote the maturation and stimulation of osteoclasts in vivo. Co-administration of E2 abrogated the adverse effects by gp46-197. This is a novel mechanism of osteolysis directly induced by HTLV-1 structural protein. We presume the therapeutic potential of E2 for the treatment of hypercalcemia in ATL patients.