ANP32A Knockout Cell Lines

Investigate the function of ANP32A (Acidic Nuclear Protein 32A) in the replication and mammalian adaptation of influenza A viruses, particularly its role in driving the acquisition of critical adaptive mutations like PB2 E627K in H7N9 avian influenza viruses.

1. Influenza Virus Host Adaptation

Avian influenza viruses (AIVs) must adapt to mammalian hosts to enable efficient replication and transmission. The PB2 E627K mutation is a key adaptive marker in AIVs, enhancing polymerase activity and pathogenicity in mammals . H7N9 viruses, which emerged in 2013, readily acquire PB2 E627K in human infections, but the underlying mechanism remains unclear .

2. Role of Host Factors in Virus Adaptation

Host proteins like ANP32A play critical roles in virus-host interactions. ANP32A is essential for influenza virus polymerase function, but its specific role in mediating PB2 E627K acquisition during AIV adaptation is underexplored .

3. Gaps in Knowledge

How does ANP32A influence influenza virus polymerase activity and adaptation?

Can ANP32A knockout (KO) cell lines serve as a model to study virus-host interactions and adaptive mutations?

1. Generation of ANP32A KO Cell Lines

Method: CRISPR-Cas9 genome editing was used to disrupt the ANP32A gene in human A549 lung cells and HEK293T cells. KO was confirmed by western blotting and sequencing, verifying the absence of ANP32A protein expression .

Validation: ANP32A KO cells showed impaired replication of H7N9 viruses with wild-type PB2 (E627), while supporting normal replication of PB2 E627K mutant viruses .

2. Functional Assays in ANP32A KO Cells

Polymerase Activity: Minigenome assays revealed that ANP32A depletion significantly reduced the polymerase activity of H7N9 viruses with wild-type PB2 (E627) in HEK293T cells, but had minimal effect on PB2 E627K mutant viruses .

Virus Replication: H7N9 virus titers in ANP32A KO A549 cells were reduced by >90% compared to wild-type cells, while PB2 E627K mutant viruses replicated efficiently in KO cells .

Adaptive Mutation Acquisition: In wild-type mice, H7N9 viruses readily acquired PB2 E627K, but in Anp32a-/- mice, virus replication was impaired, and instead, the alternative PB2 D701N mutation emerged .

3. Mechanistic Studies

ANP32A-Polymerase Interaction: GST pulldown assays showed that ANP32A interacts more strongly with H7N9 polymerases containing PA mutations that enhance polymerase activity (e.g., PA142R-147V-171V-182L), reducing the need for PB2 E627K .

Signaling Pathway Analysis: Depletion of ANP32A impaired the activation of Akt and NFκB pathways in virus-infected cells, linking ANP32A to viral signaling during adaptation .

1. Mechanistic Insights

ANP32A regulates influenza virus adaptation through two key mechanisms:

Polymerase Support: ANP32A specifically enhances the activity of avian-like polymerases (PB2 E627), driving the need for PB2 E627K to compensate for low activity in mammalian cells .

Adaptive Pathway Switching: In the absence of ANP32A, virus replication is impaired, forcing the virus to acquire alternative adaptive mutations (e.g., PB2 D701N) to maintain fitness .

2. Translational Significance

Virus-Host Interaction Modeling: ANP32A KO cell lines recapitulate critical aspects of influenza virus adaptation, enabling studies of how host factors drive viral evolution .

Therapeutic Targets: ANP32A represents a potential host-targeted therapeutic target, as its depletion impairs virus replication without affecting PB2 E627K mutant viruses, guiding the design of broad-spectrum antivirals .

3. Product Utility

ANP32A KO cell lines offer unique value for:

Investigating the role of host factors in influenza virus adaptation and pathogenicity.

Screening compounds that disrupt virus-ANP32A interactions to block adaptive mutations.

Modeling pandemic influenza scenarios and evaluating vaccine candidates.