The Mechanism Research Of Spike Protein Conformational Change Mediated By PH In Murine Hepatitis Virus

Coronavirus (CoV) is an enveloped plus-stranded RNA virus with the largest genome among all known RNA viruses. CoVs infect varieties of species including humans, other mammals and birds, and cause different diseases. Since 2003, there have been two global outbreaks of CoVs, Severe Acute Respiratory Syndrome virus (SARS-CoV) in 2002-2003 with case fatality ratio (CFR) at 9.6%, and ongoing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) since 2012 with CFR at 36.5%. Currently, there is no approved drug or vaccine treatment of CoV infection, indicating the urgency for CoV research. CoV enters cells through the interaction of the spike protein (S) with its cognate receptor. S protein is a trimer and comprised of two subunits, S1 and S2. While S1 is responsible for receptor recognition and binding, S2 contains all fusion mechinary, including a fusion peptide (FP), two heptad repeats (HR) and a transmembrane domain (TMD). Native S protein is believed to be in a metastable state. Once it binds to the receptor, it triggers series of conformational changes, leading to dissociation of S1, insertion of FP into target membrane, formation of six helix bundle by HRs, and membrane fusion. However, it remains unclear how receptor binding triggers conformational changes in S1.Mouse hepatitis virus (MHV) is a beta-CoV and considred as the model CoV. Previous studies found that S protein of MHV A59 strain mediates receptor independent syncytia (RIS) at pH8.0 effectively and conformational changes of S protein induced by pH8.0 are very similar to those triggered by binding to its cognate receptor mCEACAM1a. Therefore, it provides us a simple and nature model for studying how receptor binding triggers the conformational changes in S1 subunit.In this study, we determined the specific molecular mechanism of conformational changes of S protein induced by pH8.0 in the absence of the receptor. Histidine is the only amino acid with a pKa in the vicinity of 6.5, previous studies found that one or more histidine act as pH sensors for influenza HA protein and are vital for HA mediated membrane fusion. To investigate if histidine plays a role in MHV S protein mediated RIS, we mutated all 14 histidines in MHV A59 S protein.We found that the histidines at the position of 179,209,441,643 and 759 of spike protein are critical for S protein mediated RIS triggered by pH. We further determined if the aromatic ring in histidine is essential for its function, H179,H209,H441,H643 and H759 in S protein were mutated to phenylalanine individually,we found that aromatic ring of H759 is importannt for spike protein mediating RIS induced by pH. Since phenylalanine is not affected by pH, H759 of spike is not as a pH sensor.In summary, we found that the histidines at the position of 179,209,441,643of spike protein played important roles in pH8.0 inducing RIS, and the aromatic ring of H759 is also important for the process of pH8.0 inducing RIS.