Learning How SARS Spikes Its Quarry

TheSARS (severe acute respiratory syndrome) coronavirus was responsiblefor a worldwide outbreak in 2002-2003 that affected more than 8,000people and killed 774 before being brought under control. Public healthexperts worry about another outbreak of the virus, which originates inanimals such as civet cats.

The research team, led by HowardHughes Medical Institute investigator Stephen C. Harrison at Children'sHospital and Harvard Medical School, and colleague Michael Farzan, alsoat Harvard Medical School, reported its findings in the September 16,2005, issue of the journal Science. Lead author Fang Li in Harrison'slaboratory and Wenhui Li in Farzan's laboratory, also collaborated onthe study.

According to Harrison, prior to these studies,researchers knew that one of the key steps in SARS infection occurswhen the virus's spike protein attaches to a receptor on the surface oftarget cells. Attachment of the spike protein permits the virus to fusewith a host cell and inject its RNA to infect the cell.

Adetailed understanding of how the spike protein complexes with itsreceptor, ACE2 (angiotensin-converting enzyme 2), could have importantclinical implications. “The interest in understanding this complex hasto do with the fact that this virus jumps from animals to humans,laterally among humans, and in some cases from animals to humans butwithout subsequent human-to-human transmission,” said Harrison. “And weknow that those modes of transmission depend on specific mutations inthe spike protein that affect spike-receptor interaction.

“One ofthe critical issues in a SARS epidemic would be to predict whether agiven variant of the virus will jump species or move laterally from onehuman to the other. Understanding the structure of this complex willhelp us understand what these mutations in the spike protein mean interms of infectivity,” Harrison said.

According to Harrison,Farzan and his colleagues laid the scientific groundwork fordetermining the structure of the spike-ACE2 complex. In 2003, Farzan'steam discovered that the ACE2 protein is the receptor for the SARSvirus. They also identified a specific fragment of the spike proteinthat is involved in viral attachment.

As a result of thosestudies, researchers in Harrison's and Farzan's laboratories couldconcentrate their efforts on creating crystals of the relevantfragments of the spike protein in complex with the ACE2 receptor. Afterthey had crystallized the protein complex, the crystals were thensubjected to structural analysis using x-ray crystallography. In thiswidely used technique, x-rays are directed through crystals of aprotein. The resulting diffraction pattern is analyzed to deduce theatomic structure of the protein or protein complex under study.

Thex-ray structure revealed that the spike protein fragment showed aslightly concave surface that fits a complementary surface on thereceptor, said Harrison. There was nothing surprising about theinteraction itself, he noted. However, the studies revealed importantnew information about two specific amino acids on the spike protein.These were the amino acids that Farzan and his colleagues hadpreviously determined to be the most critical for determining how theSARS virus adapted from infecting only civets to infecting humans.

“Bothof these critical amino acids turned out to be right in the middle ofthe interface between the spike protein and the receptor,” saidHarrison. Thus, the structure reveals details about how even smallmutations in the spike protein gene that alter the identity of aminoacids at those sites can affect the virus's ability to infect humans.Such mutations enable viral transmission by altering the shape of thespike protein, which affects how well it binds to the ACE2 receptor,explained Harrison. In particular, he said, the new structure shows howmutation at one of the two sites can enable the animal SARS virus toinfect humans, but by itself this mutation does not appear to allowsubsequent human-to-human transmission.

“The observation is thata dramatic epidemiological difference can result from what looks likean almost trivial mutation,” said Harrison. “These findings give us thebeginnings of information needed — if a new virus were isolated — tomake predictive guesses about infectivity, so that we can better giveadvance warning.”

He also noted that laboratory studies indicatethat the fragment of the spike protein they used could provide thebasis of a vaccine against SARS, since it appears to be recognized bythe immune system of the host.

In future studies, Harrison andhis colleagues plan to explore the steps that occur after the spikeprotein attaches to the receptor. The researchers know that the spikeprotein undergoes a conformational change that enables the virus tofuse with the host cell.

“When there's a conformational change,it gives you an opportunity to explore the possibility of antiviraltherapeutics,” said Harrison. “When you have two conformationalstructures, you can think about how to prevent infection by inhibitingthe transition from one state to another.”