| Autor: |
Kawano K; Division of Respirology, Rheumatology, Infectious Diseases and Neurology, Department of Internal Medicine, University of Miyazaki, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan.; Clinical Laboratory, University of Miyazaki of Hospital, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan., Hashikura Y; Clinical Laboratory, University of Miyazaki of Hospital, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan., Umekita K; Division of Respirology, Rheumatology, Infectious Diseases and Neurology, Department of Internal Medicine, University of Miyazaki, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan.; Clinical Laboratory, University of Miyazaki of Hospital, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan. |
| Abstrakt: |
To mediate intercellular communication, cells produce extracellular vesicles (EVs). These EVs transport many biomolecules such as proteins, nucleic acids, and lipids between cells and regulate pathophysiological actions in the recipient cell. However, EVs and virus particles produced from virus-infected cells are of similar size and specific gravity; therefore, the separation and purification of these two particles is often controversial. When analyzing the physiological functions of EVs from virus-infected cells, the presence or absence of virus particle contamination must always be verified. The human T-cell leukemia virus type 1 (HTLV-1)-infected cell line, MT-2, produces EVs and virus particles. Here, we validated a method for purifying EVs from MT-2 cell culture supernatants while avoiding HTLV-1 viral particle contamination. EV fractions were collected using a combination of immunoprecipitation with Tim-4, which binds to phosphatidylserine, and polymer precipitation. The HTLV-1 viral envelope protein, gp46, was not detected in the EV fraction. Proteomic analysis revealed that EV-constituted proteins were predominant in this EV fraction. Furthermore, the EVs were found to contain the HTLV-1 viral genome. The proposed method can purify EVs while avoiding virus particle contamination and is expected to contribute to future research on EVs derived from HTLV-1-infected cells. |
