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Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11
Abstract:
The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.
Insights
This study examines rotavirus glycoprotein processing in infected cells, revealing distinct oligosaccharide trimming pathways for VP7 and NCVP5 within the endoplasmic reticulum (ER). Energy-dependent steps influence glycoprotein processing and translocation.
Area of Science:
- Virology
- Cell Biology
- Glycobiology
Background:
- Rotavirus SA11 assembles in the rough endoplasmic reticulum (RER), producing two glycoproteins: VP7 (outer capsid) and NCVP5 (nonstructural).
- These glycoproteins possess high mannose N-linked oligosaccharides, suggesting localization within the ER.
- VP7 and NCVP5 are integral membrane proteins, confirmed by in vitro translation and membrane association studies.
Purpose of the Study:
- To investigate the synthesis and oligosaccharide processing of SA11 rotavirus glycoproteins (VP7 and NCVP5) in infected Ma104 cells.
- To determine the intracellular location and processing steps of these viral glycoproteins.
- To elucidate the role of energy-dependent processes in glycoprotein modification and translocation within the ER.
Main Methods:
- Infected cell cultures (Ma104) were used to study rotavirus glycoprotein synthesis.
- Pulse-chase experiments with [3H]mannose were performed to track oligosaccharide processing.
- In vitro translation with dog pancreas microsomes and treatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were employed to assess membrane association and energy dependence.
Main Results:
- Oligosaccharide processing of VP7 and NCVP5 occurred in a time-dependent manner within infected cells.
- VP7 was processed to Man8GlcNAc2, Man6GlcNAc2 intracellularly, and Man5GlcNAc2 on mature virions, while NCVP5 trimming halted at Man8GlcNAc2.
- Energy inhibition (CCCP) blocked processing of VP7 and VSV G protein at Man8GlcNAc2, but VP7 processing could resume after CCCP addition, unlike G protein, suggesting an energy-sensitive translocation step.
Conclusions:
- Alpha-mannosidase activities responsible for oligosaccharide trimming are likely located within the ER.
- Distinct oligosaccharide processing pathways exist for rotavirus glycoproteins VP7 and NCVP5.
- An energy-sensitive translocation step within the ER differentiates the processing pathways of viral glycoproteins like VP7 and cellular glycoproteins like VSV G protein.