A variety of complex sugars decorate an animal virus particle

N-glycosylation profiling of porcine reproductive and respiratory syndrome virus envelope glycoprotein 5

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Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense ssRNA virus, emerged in the late 1980’s and quickly became the most serious disease of swine worldwide. Attenuated vaccines partially or completely protect individual animals from challenge, but solidly immune herds break with PRRS for unknown reasons. Neutralization escape mutants have not been described. Mechanisms of immune protection are not definitively established. Cellular infection pathways are controversial. Juan Li, my graduate student, and I felt that part of the confusion might be due to incomplete knowledge about the virion structure, which is based mostly on similarities to other viruses or inference from RNA sequence. Juan was interested in the envelope protein carbohydrates, glycans, that were proposed to function in viral assembly and infection processes, as well as induction of and resistance to neutralizing antibodies. However, there was no specific structural information. We figured that looking at the glycans themselves would at a minimum allow for more specific hypothesis testing.

We were glycobiology novices. To overcome this challenge, Juan attended a national meeting, quickly picked up key methodological approaches and, most importantly, identified a glycan structure expert, Ron Orlando, at the University of Georgia. Juan perfected virion purification and electrophoretic separation and identification methods for envelope glycoprotein 5, the only one of four predicted glycosylated proteins that was easily visible on acrylamide gels.

Juan, with Dr. Orlando and his postodoc, Shujuan Tao,  showed by direct mass spectrometry analysis that the majority of glycan structures did not contain sialic acid. She also showed that the glycans were accessible to receptor proteins. Our findings helped to explain PRRSV infection of cells lacking sialoadhesin and provided a glycan database to facilitate molecular structural studies of PRRSV. This structure study was accompanied by a function paper (Virology, 2015, 477:82-88) with the most interesting observation being that growth of PRRSV in cells treated with swainsonine to inhibit complex glycan formation, thus removing terminal structures, had no effect on viral growth and infectivity. We think that the main purpose of glycans is to facilitate processing through the Golgi.

It was thrilling to directly interrogate biological structures, an animal virus in this case, even though we lacked technical expertise. It was disappointing that months of work to more precisely map glycan structures to individual amino acids gave inconclusive results, with no opportunity to repeat the experiment. Nevertheless, learning something new every day and discovering new knowledge make science exciting.

Gylcan ms

Picture caption
Electrospray ionization spectrum shows PRRSV envelope glycoprotein 5 glycans of various molecular weights, circled in red. Fragmentation and mass analysis of two individual peaks gave molecular masses that could only arise from four or six possible structures, as indicated.

Introducing the Authors

Authors

From left to right: Juan Li, Shujuan Tao, Ron Orlando, Michael P. Murtaugh

About the research
N-glycosylation profiling of porcine reproductive and respiratory syndrome virus envelope glycoprotein 5
Virology, Volume 478, April 2015, Pages 86–98
Juan Li, Shujuan Tao, Ron Orlando, Michael P. Murtaugh

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