Related Experiment Videos
Redundant proteolytic activation of a viral superantigen
1Wadsworth Center, New York State Department of Health, Albany 12201-2002, USA.
Abstract:
Proteolytic activation of viral superantigens (vSAgs)4 expressed in Chinese hamster ovary (CHO) cells is required for T cell stimulation, and is mediated primarily by the protein convertase (PC) furin. Three PC recognition sites are highly conserved in vSAgs, but it was not known which sites are required for PC dependent vSAg activation. Moreover, because the PC recognition sites are not conserved in all functional vSAgs it was possible that activation could occur by processing at any of several sites. To identify the location(s) where processing of vSAg7 generates an active superantigen, each of two PC recognition sites, and a third related site were altered by in vitro mutagenesis, and the mutant proteins were tested for their abilities to activate T cells. Mutation of the PC recognition site at position 68-71 in vSAg7 had no effect on its ability to activate T cells. Mutation of the processing site at position 169-172 completely abolished T cell activation, and indicated that cleavage at this position was obligatory for proteolytic activation of vSAg7. However, introduction of a PC recognition site at position 192-195, a position that in many other vSAgs encodes a PC recognition site, restored activity to a vSAg7 protein that lacked a recognition site at position 169-172. The data revealed that processing of vSAgs at either position 169-172 or 192-195 was sufficient for vSAg7 activation, and explain how vSAgs that lack some PC recognition sites can be activated by proteolytic processing.
Insights
Proteolytic activation of viral superantigens (vSAgs) requires processing by protein convertases (PCs). Cleavage at position 169-172 is essential for vSAg7 activation, while other sites also enable T cell stimulation.
Area of Science:
- Immunology
- Virology
- Molecular Biology
Background:
- Viral superantigens (vSAgs) require proteolytic activation for T cell stimulation.
- Protein convertases (PCs), particularly furin, mediate vSAg activation.
- The specific PC recognition sites essential for vSAg activation were not fully understood.
Purpose of the Study:
- To identify the critical processing sites responsible for the proteolytic activation of viral superantigen 7 (vSAg7).
- To investigate the role of conserved and variable PC recognition sites in vSAg activation.
- To elucidate the mechanisms by which vSAgs with diverse recognition sites achieve functional activation.
Main Methods:
- Site-directed mutagenesis was used to alter specific PC recognition sites within vSAg7.
- Mutant vSAg7 proteins were expressed in Chinese hamster ovary (CHO) cells.
- The ability of mutant vSAg7 proteins to activate T cells was assessed.
Main Results:
- Mutation of the PC recognition site at position 68-71 did not affect vSAg7 T cell activation.
- Mutation of the processing site at position 169-172 abolished T cell activation, indicating obligatory cleavage.
- Introducing a PC recognition site at position 192-195 restored activity in a vSAg7 mutant lacking the 169-172 site.
Conclusions:
- Proteolytic processing at position 169-172 is obligatory for vSAg7 activation.
- Processing at either position 169-172 or 192-195 is sufficient for vSAg7 activation.
- This explains how vSAgs lacking certain PC recognition sites can still be activated.