Optimized method for isolation of immature intracytoplasmic retroviral particles from mammalian cells

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F17%3A00480898" target="_blank" >RIV/61388963:_____/17:00480898 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60461373:22330/17:43913335

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.jviromet.2017.06.003" target="_blank" >http://dx.doi.org/10.1016/j.jviromet.2017.06.003</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jviromet.2017.06.003" target="_blank" >10.1016/j.jviromet.2017.06.003</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Optimized method for isolation of immature intracytoplasmic retroviral particles from mammalian cells

Popis výsledku v původním jazyce

To biochemically and structurally characterize viral intracytoplasmic particles (ICAPs), a sample of high purity and homogeneity is usually required. Production of ICAPs in the system closely related to their natural host cells is crucial for the analysis of host-cell binding proteins involved in ICAPs assembly, transport and budding. However, this approach is often hampered by problems with low yield of the ICAPs due to either low expression or fast release from the host cell. Another obstacle may be a low stability or fragility of the intracellular particles. The published methods for ICAPs isolation often involved several time-consuming centrifugation steps yielding damaged particles. Other papers describe the ICAPs production in non-natural host cells. Here, we optimized the method for purification of unstable Mason-Pfizer monkey virus (M-PMV) ICAPs from non-human primate derived cells, commonly used to study MPMV replication i.e. African green monkey kidney fibroblast cell line (COS-1). Our simple and rapid procedure involved separation of the intracytoplasmic particles from the cell debris and organelles by differential, low-speed centrifugation, their purification using sucrose velocity gradient and final concentrating by low-speed centrifugation. Importantly, the method was established for unstable and fragile M-PMV intracytoplasmic particles. Therefore, it may be suitable for isolation of ICAPs of other viruses.

Název v anglickém jazyce

Optimized method for isolation of immature intracytoplasmic retroviral particles from mammalian cells

Popis výsledku anglicky

To biochemically and structurally characterize viral intracytoplasmic particles (ICAPs), a sample of high purity and homogeneity is usually required. Production of ICAPs in the system closely related to their natural host cells is crucial for the analysis of host-cell binding proteins involved in ICAPs assembly, transport and budding. However, this approach is often hampered by problems with low yield of the ICAPs due to either low expression or fast release from the host cell. Another obstacle may be a low stability or fragility of the intracellular particles. The published methods for ICAPs isolation often involved several time-consuming centrifugation steps yielding damaged particles. Other papers describe the ICAPs production in non-natural host cells. Here, we optimized the method for purification of unstable Mason-Pfizer monkey virus (M-PMV) ICAPs from non-human primate derived cells, commonly used to study MPMV replication i.e. African green monkey kidney fibroblast cell line (COS-1). Our simple and rapid procedure involved separation of the intracytoplasmic particles from the cell debris and organelles by differential, low-speed centrifugation, their purification using sucrose velocity gradient and final concentrating by low-speed centrifugation. Importantly, the method was established for unstable and fragile M-PMV intracytoplasmic particles. Therefore, it may be suitable for isolation of ICAPs of other viruses.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10607 - Virology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2017

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Virological Methods

ISSN

0166-0934

e-ISSN

—

Svazek periodika

248

Číslo periodika v rámci svazku

Oct

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

7

Strana od-do

19-25

Kód UT WoS článku

000412616900003

EID výsledku v databázi Scopus

2-s2.0-85021123879