Purification, Identification And Structure Analysis Of Hemagglutinin Protein Of An Avian H1N1 Influenza Virus

Influenza A viruses are RNA viruses that contain eight negative-sense and segmented RNA which encode eleven proteins. Hemagglutinin (HA), one of the major surface proteins of the influenza virus, is responsible for virus attachment to the receptor of host cells to initiate an infection. HA is considered to be associated with preferential specificity for receptors. Many studies show that avian viruses preferentially recognize Siaα2,3Gal receptors, while human viruses prefer the Siaα2,6Gal receptors. This can account for the limited replication of avian virus in human and human viruses in avian. Since pig trachea contains two receptor types, it serves as"mixing vessels"for avian and human viruses. When avian virus acquire the ability of binding Siaα2,6Gal receptors in pig, it can transmit to human. However, the H5N1 avian influenza virus that caused an outbreak in HK in 1997 has the 2,3 receptor binding specificity. So receptor specificity may not be as stringent as previously recognized in some avian viruses.H1N1 has a long history of human infection, and it circulates in human, swine and avian hosts.It is an ideal model for studying host adaptation and cross species transmission of influenza. Therefore, we choose an avian H1N1 virus of A/WDK/JX/12416/2005 as research strain. In our study, we purified the HA by ultracentrifugation, sucrose gradient density centrifugation, ion exchange, SDS-PAGE electrophoresis, and so on. This is a protocol to directly acquire the HA from virus particles, it make sure the HA structure is in native state.In our study, nanoelectrospray MS/MS was used to identify the purified HA. When compare the sequence of peptide with the HA gene, we found seven variations between HA protein and gene, namely E103K, R130K, T169I, I338V, N387S, S398I/L, and I399S. Because influenza virus uses different polymerase machineries for replication and transcription, these substitutions could be introduced in the viral genome through replication process or in viral mRNA in the transcription. The results, for the first time, provided experimental evidence showing differences in AA sequence obtained from direct analysis of viral protein derived from viral genome.We determined the structure of HA, as well as its complexes with receptor analogs by X-ray crystallography in our study. The results show that the HA structure is similarly between avian and mammal influenza virus except the glycosylation mode. However, the structure of complexes revealed no preferential binding of avian receptor analog over that of the human analog. That is to say, HA show the same binding efficiency to two receptor types. Therefore, glycosidic linkage of receptor may not the discriminating factor in virus recognition of receptor type in some subtypes of influenza viruses, such as the H1N1 viruses.