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Published on: January 28, 2019
Characterization and Transferring of Human Rotavirus Double-Layered Particles in MA104 Cells
Ali Teimoori1, Hoorieh Soleimanjahi1, Manoochehr Makvandi2
1Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
Background:
Rotavirus (RV) is a major cause of gastroenteritis in infants and children and is one of the most severe public health problems. Rotaviruses outer layer contains two proteins including VP4 and VP7. These proteins are necessary for host-cell binding and penetration. TLP (triple layer virus particle) of RV is a complete infectious virion that binds to the target cells and internalized at the cytoplasm. The DLP (double layer virus particle) is a non-infectious particle that is formed through exclusion of the outer layer proteins including VP4 and VP7. These DLPs are the transcriptionally active forms of rotavirus.
Objectives:
The aim of this study was to transfer DLP of RV into cytoplasm of MA104 cells by Lipofectamine and to analyze their replication.
Materials And Methods:
Initially, rotavirus was purified by CsCl discontinuous gradient and DLP was separated from TLP based on density differences. For confirmation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins were conducted Then the purified DLP of RV was transferred into MA104 cells using Lipofectamine.
Results:
We attempt to avoid the attachment and entry of the rotavirus by using Lipofectamine to mediate the delivery of viral particles directly into the cytoplasm. DLP was endocytosed into the cytoplasm following treatment by Lipofectamine and then replicated in cytoplasm.
Conclusions:
Therefore the non-infectious DLPs were became infectious if introduced into the cytoplasm of permissive and cancerous cells, without passing attachment and entry process.
Insights
Non-infectious rotavirus double-layer particles (DLPs) became infectious after direct cytoplasmic delivery into MA104 cells using Lipofectamine. This bypasses the need for viral attachment and entry processes for replication.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Rotavirus (RV) causes severe gastroenteritis in children, posing a significant public health challenge.
- RV's outer proteins (VP4, VP7) mediate cell binding and entry; TLP is infectious, while DLP lacks these outer proteins.
- DLPs are transcriptionally active but non-infectious due to the absence of outer layer proteins.
Purpose of the Study:
- To investigate the direct cytoplasmic delivery of RV DLPs into MA104 cells using Lipofectamine.
- To analyze the replication of DLPs following successful cytoplasmic transfer.
Main Methods:
- Rotavirus was purified, and DLPs were separated from TLPs using CsCl gradient centrifugation.
- Protein composition was confirmed via SDS-PAGE.
- Lipofectamine mediated the delivery of purified DLPs into MA104 cells.
Main Results:
- Lipofectamine facilitated the endocytosis of DLPs directly into the cytoplasm, bypassing normal viral entry pathways.
- Delivered DLPs successfully replicated within the cytoplasm of MA104 cells.
Conclusions:
- Non-infectious rotavirus DLPs can be rendered infectious upon direct introduction into the cytoplasm of permissive cells.
- This method circumvents the requirement for viral attachment and entry, offering a novel approach to studying RV replication.
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