Regulation of influenza virus RNA synthesis machinery by viral and host factors

Thesis

Influenza A virus transcribes and replicates the viral genome in the context of ribonucleoproteins (RNPs) which are the basic RNA synthesis units of the virus. The function of this viral RNA synthesis machinery is tightly regulated by various viral and host proteins to ensure the optimized virus replication and concomitant evasion or subversion of host immune responses.

ANP32 is recognized as an essential host factor for influenza virus replication due to its role in mediating the formation of influenza viral polymerase asymmetric dimer. In this work, we unravel that ANP32 facilitates recruitment of NP to the nascent RNA products, which is the second function of ANP32 during influenza virus replication, further strengthening the model that FluPol-ANP32 complex represents the replication platform for the influenza virus RNA genome.

Influenza virus nucleoprotein (NP) is the major component of ribonucleoproteins, and it binds to viral RNA and interacts with viral polymerase, underlying its critical role in virus replication. I demonstrate that the C-terminal tail of NP, a region that was previously only characterized by structural analysis, is important for cRNA stabilization and FluPol-NP interactions. Mutation in the C-terminal tail of NP profoundly attenuates the virus, recapitulating the essential role of NP in influenza virus replication. Another important viral regulator of the viral RNA synthesis machinery is the influenza nuclear export protein (NEP). I establish a split-luciferase assay to measure FluPol-NEP interactions. Based on our resolved structure of FluPol in complex with NEP, we identify critical residues at the interface of the FluPol-NEP complex and link the FluPol-NEP interactions with the regulatory function of NEP.

The interdependent nature of vRNA and cRNA synthesis of influenza virus hinders the understanding of the effect of different factors on each step of virus replication. By combining the RNPs isolation techniques and in vitro vRNP reconstitution system, we set up an in vitro cRNP reconstitution assay, which allows us to uncouple the vRNA and cRNA synthesis and measure the authentic vRNA synthesis from isolated cRNPs alone.

In summary, the above findings improve our understanding of mechanisms by which ANP32, NP, and NEP regulate influenza virus RNA synthesis. The techniques established in this study also provide powerful tools for future studies on the replication mechanisms of influenza virus.

Authors: Wang, F

• DOI: 10.5287/ora-dpkkkbgr6
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: RNA-dependent RNA polymerase; viral RNA synthesis; Influenza virus
• Subjects: Molecular virology; Virology