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University of Manitoba Proteomics Researchers Tackle SARS Virus Proteins


Last week, as two teams of researchers in Canada and the US drew worldwide attention for their completed draft sequences of the severe acute respiratory syndrome coronavirus genome, proteomics researchers were quietly working their end of the SARS problem.

At the Manitoba Proteomics Centre, John Wilkins, Kenneth Standing, and their colleagues sequenced two of the coronavirus proteins by mass spectrometry, one of which appears to be recognized by the immune system and could potentially become the starting point for a vaccine.

It all started about two weeks ago, said Wilkins, who is co-director of the Manitoba Proteomics Centre at the University of Manitoba, and a professor in the department of immunology. That’s when Frank Plummer, scientific director of Canada’s National Microbiology Laboratory in Winnipeg, asked him if the center could identify proteins found in supernatants of virus cultures originating from a SARS patient — the same material that was sent to the British Columbia Cancer Center for genome sequencing. The microbiologists had observed by western blotting that one of these proteins was recognized by sera from several SARS patients who had recovered from the disease, suggesting that the protein might elicit an immune response.

Wilkins agreed to help with the identification, and his lab prepared the proteins for de novo sequencing by mass spectrometry, which was conducted in the Time-of-Flight Laboratory headed by Kenneth Standing and Werner Ens, using a prototype of the Sciex Q-STAR tandem quadrupole TOF instrument that this group developed. De novo sequencing was necessary, said Wilkins, because the protein sequence did not show significant homology to sequences in the database. According to Standing, the scientists were successful at sequencing one protein almost completely, and another, less abundant one to a large extent. The researchers are now preparing their results for publication, and while they could not reveal the identity of the proteins, Wilkins said that the immunoreactive one is “probably not the one you would have most expected."

According to Susan Weiss, a professor of microbiology at the University of Pennsylvania, the open reading frames predicted by the SARS virus genome show homology to four proteins found in a murine coronavirus that she has been studying. These proteins comprise a replicase, a nucleocapsid protein, and two glycoproteins called Spike and membrane or M protein. Spike is the main attachment protein of the mouse virus.

Both proteins sequenced by the Manitoba researchers also turned out to match the BC Cancer Center’s coronavirus genome sequence, which was helpful to resolve ambiguities in the protein sequence: “At the time they did [the genome], we already had the mass spec data, we had all the pieces of the jigsaw puzzle,” said Standing. However, the genome sequence provided the full picture, “and as you know from making jigsaw puzzles, it’s an awful lot easier to put things together if you know what the picture should look like,” he said.

The Manitoba researchers’ results are significant for a number of reasons. For a start, while Steven Jones and his team at the BC Cancer Center have predicted 11 ORFs from the genome sequence they assembled, this analysis actually identified two SARS virus proteins that are expressed. Not only that, one of these proteins also seem to be involved in the body’s immune response, as a majority — albeit not all — of the patient sera reacted with it. What remains to be proven, Wilkins said, is whether the sera recognize the pure protein.

The protein could also be a possible starting point for a vaccine or a drug. “This [study] is somewhat a long way before that. But if you are going to make something like a vaccine, you have to know what to target. This would seem to be a possible target,” said Standing.

The Manitoba researchers might well be involved in further analyses of SARS virus proteins. They have already collaborated with the National Microbiology Laboratory on previous occasions, said Wilkins, and are available to do so again. “This may be the tip of the iceberg; it’s hard to tell,” said Standing. “We put enough effort into it so far that obviously, if there are more experiments that need to be done, we will try to do them.”

— JK


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