117th lab seminar Ho-Su Shin

1. Human endogenous retrovirus(HERV-K) particles in megakaryocytes cultured from essential thrombocythemia peripheral blood stem cells 117th lab seminar Ho-Su Shin

2. Objective The aim of this study was to determine the extent of human endogenous retrovirus (HERV) gene trans –lation in megakaryocytes cultured from peripheral blood stem cells of patients with essential thrombocythemia previously reported with platelet-associated HERV sequences and reverse transcriptase activity. Molecular biology & Phylogeny Laboratory

3. Fig 1. Pluripotent stem cell lineage Molecular biology & Phylogeny Laboratory

4. HERV-K The HERV-K family of endogenous elements have still biological activity 1. HERV-K sequences have been detected in the genomes of great apes and Old World monkeys, New World monkeys do not possess this family of sequences 2. Most HERV-K members are transcriptionally active and, unlike many of the other sequenced HERV elements, the prototype HERV-K10 provirus contains very few mutations that interrupt the viral open reading frames Molecular biology & Phylogeny Laboratory

5. 3. HERV-K elements encode the retrovirus-like particles produced by the teratocarcinoma cell line GH and the particles detected in the placenta of primates Molecular biology & Phylogeny Laboratory

6. Fig. 2. Phylogenetic analysis of the HSRV K group by the neighbour-joining method. Molecular biology & Phylogeny Laboratory

7. Fig 3. Ratios of synonymous to nonsynonymous substitutions (ds/da) in HERV-K10-related sequences. Molecular biology & Phylogeny Laboratory

8. Figure 2 Expression of HERV-K10-like gag gene in leukemia samples and normal PBMCs. Molecular biology & Phylogeny Laboratory

9. Material & Method Terminally differentiated megakaryocytes derived from circulating stem cells in serum-free medium supplemented with stem cell factor and thrombopoietin were processed for electron microscopic immunostaining using a monoclonal antibody against the gag protein of HERV-K10 and an electron dense gold-labeled secondary antibody. Molecular biology & Phylogeny Laboratory

10. Results We found that HERV-K gag protein was detected as clusters in the cytoplasm as well as associated with viral particles budding from the cell membrane and into intracellular vacuoles in megakaryocytes from two patients with essential thrombocythemia. None of these structures was observed in megakaryocytes from a normal control or from a patient with chronic myelocytic leukemia. Molecular biology & Phylogeny Laboratory

11. Figure 1. Megakaryocytes cultured for 12 days from peripheral blood stem cells of patients with (A) essential thrombocythemia (ET) or (B) chronic myeloid leukemia (CML). Cytocentrifuged cells were stained with Wright-Giemsa and examined at 400× magnification. Molecular biology & Phylogeny Laboratory

12. Figure 2. (A) Electron immunolabeling of HERV-K gag protein in a megakaryocyte cultured for 12 days from peripheral blood stem cells of a patientwith essential thrombocythemia. The sites of primary monoclonal HERV-K gag antibody localization on the virus particle are visualized by an electrondense gold-labeled secondary antibody. (B) Enhanced view and higher magnification of the budding viruslike particle. Molecular biology & Phylogeny Laboratory

13. Figure 3. Cultured essential thrombocythemia megakaryocytes with immunolabeled HERV-K gag protein associated with a single virus particle budding from the cell membrane (A) and into a vacuolar space (B). Molecular biology & Phylogeny Laboratory

14. Figure 4. Essential thrombocythemia megakaryocytes with immunolabeled HERV-K gag protein associated with an extracellular viruslike particle (A), anempty cell membrane-bound particle (B), and clustered in the cytoplasm (C—E). Molecular biology & Phylogeny Laboratory

15. Conclusion This is the first evidence of HERV-K protein synthesis (gene translation) in human tissue other than seminomas, placenta, or fetal tissue. Translation of the HERV-K gag gene with subsequent packaging of the protein product into viral particles adds a new and important dimension to future studies on the role of HERVs in the myeloproliferative diseases. Molecular biology & Phylogeny Laboratory