Study on function of NS1 protein in influenza A virus replication and vaccine application of DelNS1 viruses

Abstract

Influenza A virus causes significant morbidity and excessive mortality around the world every year and has precipitated four human pandemics during the past 100 years. While significant progress has been made in influenza research since the H5N1 outbreak in Hong Kong in 1997, many questions regarding how influenza virus replication is regulated remain to be answered. It is still not clear if a universal influenza vaccine may be a realistic option for preparedness of future emerging influenza viruses which pose a pandemic threat. Influenza virus non-structural protein 1 (NS1) is a virulence determinant with multiple functions; it is the major antagonist of innate immunity against influenza A virus and also plays an important role in regulating virus replication. More significantly, influenza A virus with NS1 gene deleted is highly attenuated and DelNS1 virus is being actively explored as a new live attenuated vaccine for both seasonal and pandemic influenza. To better understand the role of NS1 in the influenza A virus life cycle, I established three DelNS1 viruses from different strains to study the regulation of influenza virus replication and assess their potential for vaccine development.

I found that the WSN-DelNS1, H7N9-DelNS1 and CA04-DelNS1 virus each acquired an M-A14U, M-G917A and NP-G346A mutation through passages either in MDCK, Vero or 10-day-old chicken embryonated eggs. Characterization of growth properties found that these three DelNS1 viruses replicated to a level comparable to the wild type (WT) virus in each system. This is by far the most direct evidence to indicate that the NS1 protein is dispensable for influenza virus replication. Further analysis using WSN-DelNS1-M-WT virus revealed that the M2 expression was markedly attenuated due to the loss of NS1 expression, and M-A14U mutation restored M2 expression during virus replication. My findings clearly demonstrate a mechanism that both NS1 and viral polymerases are involved in the regulation of M mRNA splicing during virus replication. I further characterized WSN-DelNS1-M-A14U virus and found that the M-A14U mutation increased the M2 mRNA splicing by inhibiting the mRNA3 splicing via impairment of the mRNA3 splicing consensus sequence. The role of NS1 in regulation of M mRNA splicing is further confirmed using the H7N9-DelNS1-M-G917A virus.

Prior to this study, there were no reports evaluating the vaccine applications of adapted replication competent DelNS1 virus. I found DelNS1 viruses to be completely avirulent to mice at the highest available dose. Most importantly, this study found that DelNS1 H1N1 viruses are able to provide cross protection against lethal challenges of heterosubtypic viruses, including highly pathogenic avian H5N1 and H7N9 viruses. The DelNS1 viruses developed in this study provide valuable tools warranting evaluation for use as universal vaccines to control and prevent emergence of future influenza virus strains.

In summary, this study provides direct evidence showing that NS1 is involved in influenza virus replication through regulating the splicing of M gene, and establishes a strategic technology for developing DelNS1 live attenuated vaccines.