Mechanisms by which NS1 and NEP govern the influenza lifecycle

About This Grant

The influenza virus (IAV) is a serious public health concern causing seasonal epidemics and sporadic pandemics with significant mortality and morbidity worldwide. Yet, there are several basic aspects of the viral lifecycle that remain under-investigated from a mechanistic and structural perspective. Filling these gaps in knowledge would not only yield fundamental knowledge of influenza biology, but it would also identify novel targets for the development of critically needed antivirals against influenza. Considering their critical roles in the influenza lifecycle, the influenza non-structural protein 1 (NS1) and nuclear export protein (NEP) are the focus of this proposal as they have been highlighted in numerous studies as viable targets for the development of antiviral therapeutics. NS1 is a virulence factor that suppresses the innate immune response to infection by interacting with both nuclear and cytoplasmic proteins, while NEP facilitates nuclear export of the vRNP complex by serving as an adaptor between the host nuclear exportin CRM1 and the vRNP complex. Furthermore, NS1 and NEP functions are predicated on their ability to translocate between the nucleus and cytoplasm of the host cell And since NS1 and NEP are encoded by the same segment of the influenza genome, mutations in NS1 are frequently accompanied by concomitant mutations in NEP and vice versa. Despite their obvious connection, most of the previous work does not focus on differentiating how individual NS1 and NEP mutations govern specific steps of the influenza lifecycle. Despite our knowledge of the functions that NS1 and NEP have during the viral lifecycle, a mechanistic and structural understanding of how they accomplish their functions remain largely unknown. To fill this gap in knowledge, the objective of this application is to define the structure-function relationships that regulate both NS1 and NEP function. The rationale that underlies the proposed research is that elucidating the mechanisms by which NS1 and NEP govern the influenza lifecycle. Furthermore, defining these structure- function relationships will provide detailed structural analysis for the development of novel influenza antivirals that target NS1 and/or NEP function. We will achieve our objective by pursuing three specific aims: 1) structurally characterize the multiple interactions between NS1 and host proteins previously implicated in the regulation of NS1 intracellular transport, 2) solve the high-resolution structure of NEP in complex with the CRM1 export machinery, and 3) determine how these interactions affect various aspects of the viral lifecycle such as replication kinetics and pathogenicity. Our innovative approach will combine several state-of-the-art techniques to perform studies that span from protein structural studies to live cell imaging of NS1 intracellular distribution to in vivo pathogenesis. The proposed research is significant because it will define the molecular mechanisms that govern processes that touch on a number of fundamental aspects of influenza biology. By defining these molecular mechanisms, this proposal will inform efforts in developing influenza antiviral drugs that target NS1, thereby supporting the overall mission of the NIH.