Engineering influenza resistance: assessing ANP32-edited chicken cells
Ewa Brzuchacz (student), and Dr. Carol Sheppard (supervisor), Imperial College London

Influenza Virus has a negative-sense, single-stranded, segmented RNA genome. Three of the eight segments encode for the viral proteins PB1, PB2 and PA that make up the viral polymerase (FluPol). It has been found that FluPol is strictly dependent on the avian host protein ANP32 to support replication.

ANP32 is a family of three proteins that serve native functions in hosts (A, B and E). ANP32A in chickens is the only member of the family that can support FluPol activity by two critical binding residues: 129I and 130N.

Recent studies have shown through substitution mutations ANP32AN129I-D130N introduced as gene edits into chickens meant that low dose high pathogenic IAV H9N2 was unable to replicate. However, high-dose H9N2 exposure had caused FluPol escape mutations, PAE349K and PB2M631L to utilise the other ANP32 members.

Current work by the Sheppard and Barclay lab aims to comprehensively eliminate avian host support for FluPol.

The project aimed to

• Rescue a H9N2 UDL 6:2 Bright Flu reporter virus (for the first time).
• Aid in the establishment and maintenance of stable chANP32B-mCherry and E-mCherry cell lines.
• Integrate amino acid substitutions in lentiviral plasmids expressing chANP32E-mCherry and chANP32B-mCherryClone several genes for use in NanoBit assays.

Research Highlights

• UDL H9N2 6:2-BF rescued (for the first time in the lab) for use in FACS experiments.
• Contibuted to establishing and maintaining stable chANP32B-mCherry and E-mCherry cell lines.
• Cloned PB1 and huB for NanoBit assay.

Challenges, and lessons learned

• During lentiviral site-directed mutagenesis, I started with a more difficult approach (introducing two-point mutations at once), which didn’t work.
  • Troubleshooting with different enzymes.
  • Decided to take a lower risk approach (using primers to introduce one mutation at a time).
• Troubleshooting and doing quality control (like gel electrophoresis) along the way is important – especially in cloning. Techniques like colony PCR are useful for this.

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