Discovering and characterising the cellular regulators of influenza virus RNA replication
File(s)
Author(s)
Rasmussen, Amalie Boye
Type
Thesis
Abstract
The influenza A virus (IAV) polymerase facilitates either transcription or bidirectional replication of the viral RNA genome. These distinct activities are regulated by viral proteins and by co-opted host cell proteins that stabilise the polymerase in the different complexes required for each activity. A well-characterised example is host ANP32, which is essential for viral RNA replication. Previous proteomic screens have identified many host cell proteins that support polymerase activity, but it remains unclear which direct transcription and which replication.
In this project, we aimed to identify host proteins co-opted as either transcription- or replication-specific factors. We performed a differential interactome screen by comparing the polymerase interactome in WT cells, where both viral transcription and replication take place, to the interactome in ANP32-KO cells, where replication does not occur. The IAV polymerase was shown to interact with distinct sets of host proteins for transcription and replication. Using various gene depletion methods and experimental conditions that uncouple replication and transcription, we functionally characterised HMGB2 and RUVBL2 as replication host factors and RPAP2 as a transcription host factor. We also explored the potential use of these host factors as targets for host-directed antiviral drugs.
In addition to co-opting host cell proteins as proviral factors, IAV also evades restriction by antiviral factors, such as BTN3A3 and MxA. Both are interferon-stimulated genes that restrict avian IAVs in an NP-dependent manner. We tested the BTN3A3 and MxA sensitivity of NPs from circulating avian viruses that have caused recent widespread outbreaks in birds and mammals. We also investigated the mechanism behind BTN3A3 restriction and demonstrated that BTN3A3 inhibits the cRNA synthesis step of avian IAV RNA replication.
In summary, this project has advanced understanding of the virus-host interactions that regulate the distinct activities of the IAV polymerase, leading to insights into new antiviral strategies and host restriction.
In this project, we aimed to identify host proteins co-opted as either transcription- or replication-specific factors. We performed a differential interactome screen by comparing the polymerase interactome in WT cells, where both viral transcription and replication take place, to the interactome in ANP32-KO cells, where replication does not occur. The IAV polymerase was shown to interact with distinct sets of host proteins for transcription and replication. Using various gene depletion methods and experimental conditions that uncouple replication and transcription, we functionally characterised HMGB2 and RUVBL2 as replication host factors and RPAP2 as a transcription host factor. We also explored the potential use of these host factors as targets for host-directed antiviral drugs.
In addition to co-opting host cell proteins as proviral factors, IAV also evades restriction by antiviral factors, such as BTN3A3 and MxA. Both are interferon-stimulated genes that restrict avian IAVs in an NP-dependent manner. We tested the BTN3A3 and MxA sensitivity of NPs from circulating avian viruses that have caused recent widespread outbreaks in birds and mammals. We also investigated the mechanism behind BTN3A3 restriction and demonstrated that BTN3A3 inhibits the cRNA synthesis step of avian IAV RNA replication.
In summary, this project has advanced understanding of the virus-host interactions that regulate the distinct activities of the IAV polymerase, leading to insights into new antiviral strategies and host restriction.
Version
Open Access
Date Issued
2025-09-08
Date Awarded
2026-02-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Barclay, Wendy
Sponsor
MRC DTP
Grant Number
G98687
Publisher Department
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)