Role Of HTLV-1HBZ In Regulation Of Cyclin D1Expression And Establishment Of ELISA Assay For HTLV-1

Background and objective:Human T-cell lymphotropic virus (HTLV) was the first retrovirus discovered in humans. HTLV-1and HTLV-2share approximately70%homology at genetic level. The two major pathologies associated with HTLV-1infection and present in all endemic areas are:adult T-cell leukemia (ATL) and HTLV-1associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1/2infections can be transmitted by vertical route, sexual intercourse and parental (blood transfusion and intravenous drug use), and spread globally. HTLV-1is endemic in regions of the Japan, Caribbean, Central and Western Africa, South and Central America. HTLV-2has more prevalent among some native Americans and some Central African tribes, but is relatively common among some special population in Europe, North America, and other regions of the world. China is considered to be a non-endemic region for HTLV. Since1985, several investigations of the prevalence of HTLV have been carried out in China and the prevalence rate0.06%to1.27%. The highly endemic areas for HTLV were located in the Eastern coastal region of China. Little information on HTLV-1/2prevalence has been found in Henan and Hubei province, which with large and great migrant population. However, the methods used for detected HTLV-1/2infections have some limitations, because of both viruses are endemic and where HTLV viral isolates could diverge from the isolates.Recently, a novel viral protein HTLV-1bZIP factor (HBZ), which is encoded by an mRNA of the opposite polarity of the viral genomic RNA, has been identified in HTLV-1infected cells. HBZ was found to be expressed in all ATL cases and can inhibit Tax-mediated transactivation of viral transcription. HBZ, via its bZIP domain, also interacts with the host’s bZIP factors, thereby modulating their transcriptional activity. These findings suggest that HBZ is a critical factor in leukemogengsis. But the mechanisms of HBZ on inleukemogenesis of HTLV-1infected cells were still unknown.Our study focused on HBZ and discussed the effects of HBZ on cyclin Dl expression; initial established an ELISA assay for HTLV-1; take an epidemiological investigation of HTLV-1and HTLV-2Infection among different population in Central China. These results provide a theoretical basis for the role of HBZ on cell cycle transition, improve the screening methods, and know the status of HTLV infection in Henan and Hubei province.Chaper I The HTLV-1HBZ protein interacts with the cellular transcription factor CREB inhibit cyclin D1expressionMethods1. Full-length HBZ and HBZ-AD, HBZ-bZIP were generated by PCR amplification. These fragments were cloned into the lentiviral vector (LV5). Then the lentiviral vectors were packed and the virus were harvested and concentrated.2. Using RT-PCR and Western Blot to detect the cyclin D1mRNA and protein expression level in293T-HBZ cells、CEM-HBZ cells and Jurkat-HBZ cells.3. Construct the cyclin Dl promoter deletion mutants and detect the cyclin D1promoter activity in different HBZ expressing cells.4. Total protein was extracted from239T cells and293T-HBZ cells. Precleared cell lysates were incubated with anti-HA and immunocomplexes were analyzed by Western Blotting with anti-CREB antibody.5. Construct the expression plasmid pGEX-2T-CREB. GST pull-down experiments were performed using purified GST fusion protein and HBZ protein to detect the interaction of HBZ with CREB in vitro.6. Construct the expression plasmid pGEX-2T-HBZ-bZIP. GST pull-down experiments were performed using purified GST fusion proteins to detect the interaction of HBZ-bZIP and HBZ-AD with CREB in vitro.Results1. The sequencing and reconstriction enzymes results of three lentiviral vectors was cloned successfully; cells were transfected with lentiviral vectors and stable expressed HBZ、HBZ-AD and HBZ-bZIP.2. The results of RT-PCR and Western Blot shown, the HBZ expressing293T cells and HBZ-bZIP expressing293T cells, when compared with the empty293T cells, show reduced expression levels for cyclin D1protein. These results indicate that HBZ via its bZIP domain suppresses cyclin D1mRNA level, thereby reducing its protein expression.3. The cyclin D1promoter reporter plasmid pGL3-CD1transfected into empty CEM cells, CEM-HBZ cells, CEM-HBZ-AD cells and CEM-HBZ-bZIP cells. The cyclin Dl promoter activity decreased in HBZ and HBZ-bZIP stable expression CEM cells. When the CRE site was included (CD2, CD3), the promoter activity was not reduced; when CRE site was deleted (CD4), the promoter activity decreased to50%in empty cells and HBZ-AD cells. These results suggested that the CRE site is critical for cyclin D1promoter activity.4. The results of CoIP shown, whole-cell extracts prepared from293T-HBZ were immunoprecipitated using a anti-HA antibody, and CREB could be detect in the complex. GST pull-down assays using purified GST fusion proteins shown CREB interacts directly with HBZ in vitro.5. Further GST pull-down assays results shown, purified GST-CREB fusion proteins bind with HBZ-bZIP and purified GST-HBZ-bZIP fusion proteins could bind with CREB either; purified GST-CREB interacts with HBZ, rather than HBZ-AD domains.Chaper II Establishment of ELISA assay for HTLV-1based on HBZMethods1. The HBZ was amplified by PCR and cloned into T-vector, then subcloned into the prokaryotic expression vector pET-29a after digestion by restriction enzyme. After identified by PCR, restriction enzyme digestion and sequencing, HBZ prokaryotic expression vector was constructed.2. The expression of HBZ protein was induced by IPTG, and purified using His-tag by Ni2+affinity chromatography. After identified by SDS-PAGE electrophoresis and Western Blot, target protein was dialyzed purification further.3. Antigen of HBZ protein coated microtiter plates, avidin HRP-labeled HBZ protein was used as enzyme-labelled antigen. Double antigen sandwich method was conducted to detect antibodies in serum samples. Optimize the reaction conditions and establish HTLV-1ELISA method.4. Through the detection of standard serum and clinical serum, comparison with other commercial kits, evaluating the ELISA detection method.Results1. The HBZ fragment was amplified by PCR. The recombinant prokaryotic expression vector pET-29a-HBZ was constructed successfully through digestion of restriction enzyme and sequencing.2. SDS-PAGE electrophoresis showed that there was a specific band at25kD after IPTG induction. After protein purified by Ni2+affinity chromatography, SDS-PAGE electrophoresis showed that there was a pure band at25kD. Western Blot results showed that the purified protein has good antigenicity of HBZ.3. The enzyme labeled antigen activity of purificated HBZ protein after biotin-labelling was good through identified by an avidin. The optimum conditions of ELISA test:the reaction time of "three steps mode" were40min, the volume of serum was30μl.4. The results of test for standard serum shown this ELISA assay has good efficiency. Stability test results showed that this ELISA assay had good stability. Compared with other kits for the test of clinical serum samples shown the sensitivity and specificity were71.6%and96.7%, Kappa value was0.54, and the results were good.Chapter Ⅲ Epidemiological analysis of HTLV-1and HTLV-2infection among different population in Central ChinaMethods1. A total number of5480blood samples were collected and included into three groups, which were blood donors (3548cases), patients with malignant hematological diseases (908cases) and high risk group (1024cases). All samples were screened for the presence of HTLV-specific antibodies using an enzyme linked immunosorbent assay from Beijing Wantai Biological Pharmacy Enterprise Co.,2. One-way ANOVA and multivariate logistic regression methods were used to analysis infection status and risk factors.3. Proviral DNA was prepared form peripheral blood lymphocytes of the HTLV-1positive sample. Each major coding regions and the LTR were amplified. The PCR products were purified and purified products were cloned into the pGEM-T vector. DNA sequences were assembled and edited for complete genome. The complete genome sequence of HTLV-1was performed the phylogenetic analysis.Results1.17cases were positive for anti-HTLV antibody using the ELISA assays. Seven of17samples were confirmed positive for anti-HTLV-1antibody and three of17samples positive for anti-HTLV-2antibody using the immunoblot. 2. There was no significant difference in seroprevalence between HTLV-1and HTLV-2(P=0.21). The prevalence of HTLV-1in high risk group was significantly higher than in blood donors (P=0.03). The infection rates with HIV, HBV and HCV among HTLV-1positive individuals were significantly higher than that in HTLV-1negative individuals (P<0.05). HTLV-1positive status was associated with HIV (P=0.006) and HBV (P=0.000).3. The complete genome sequences KC807984can be recognized in the Genotype A. This new strains formed a tight clade with other genome form China and clustered with genomes from Japan and Canada.Conclusions1. HBZ repress cyclin Dl promoter activity at the CRE site through its bZIP domain, and that HBZ-bZIP reduces cyclin D1transcriptional and expression level through binding with cellular factor CREB. This observation could provide new insights into mechanisms of HBZ in cell cycle transition.2. Initial establishment of ELISA assay for HTLV-1based on HBZ.3. Epidemiological screening find that there have HTLV-1/2positive cases in Central China, the prevalence of HTLV-1/2in high risk group is comparatively higher than in blood donors and lymphoma/leukemia patients; HTLV-1positive status is associated with HIV and HBV; the new strains form a tight clade with other genome form China; our results provide theoretical basis for investigations of HTLV prevalence in specific population and revise standard of voluntary blood donation in this area.