The aim of this project is to structurally and functionally characterise the influenza virus nuclear export machinery that is responsible for the nuclear export of viral ribonucleoprotein (RNP) complexes for assembly into virions at the cell membrane. Structural and functional data about the influenza virus nuclear export protein (NEP) and its interaction with viral and cellular factors would greatly expand our limited knowledge of the mechanisms that influenza virus uses to transport its RNA genome out from the host cell nucleus.
The influenza virus genome consists of eight single stranded negative-sense RNAs that form viral ribonucleoprotein (vRNP) complexes with the viral RNA polymerase (PB1, PB2 and PA) and oligomeric nucleoprotein (NP). vRNPs display a double-helical arrangement resembling a large loop twisted into a helical filament. The RNA polymerase is responsible for replicating vRNPs in the nucleus of infected cells. The nuclear export of vRNPs is facilitated by the viral factor NEP that mediates the interaction between vRNPs and the cellular export factor Crm1/RanGTP in a viral matrix protein 1 (M1) dependent manner (see Figure). However, the structure of NEP and the molecular details of its interaction with vRNPs and Crm1/RanGTP, as well as the role or M1, remain poorly characterised at the molecular level.
Recently, we developed methods to express and purify mg quantities of NEP and obtained a preliminary structure using NMR (unpublished data). This revealed that the nuclear export signal (NES) of NEP is located on an N-terminal a-helix. Moreover, using mass spectrometry, we found a phosphorylation site in NEP (S24) that regulates its interaction with Crm1. Replacement of S24 with a phosphomimetic glutamic acid results in NEP forming stable complexes with Crm1/RanGTP. Initially, we aim to determine the structure of NEP in complex with Crm1/RanGTP using cryo-EM. However, ultimately we would like to extend these studies to complexes of vRNPs to reveal the atomic details of the interactions of the multiple viral and cellular factors involved.