Location: Plant Genetics Research
Project Number: 5070-31320-001-020-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 1, 2025
End Date: Feb 28, 2027
Objective:
Objective 1: Discovery of ANP32A/B polymorphisms across domestic swine.
Objective 2: Assess the effect of selected ANP32A/B variants on influenza A polymerase binding and activity.
Approach:
Objective 1: Discovery of ANP32A/B polymorphisms across domestic swine.
Experimental Design: We will identify ANP32A/B polymorphisms and isoforms present within domestic swine by combining targeted gene sequencing of commercially relevant purebred and crossbred sample pools using the an in-house sequencing platforms and alignment of sequencing reads from distant and ancestral breeds or wild relatives publicly available within the Sequence Read Archive. The effect of each polymorphism or alternative splicing on function and/or expression be characterized using protein sequence prediction, isoform-specific qPCR, and motif screening.
Objective 2: Assess the effect of selected ANP32A/B variants on influenza A polymerase binding and activity.
Experimental Design: We will first generate an appropriate in vitro model lacking endogenous ANP32A and ANP32B expression using CRISPR-Cas9 gene editing. Specifically, pig cell lines will be dual transfected with CRISRP-Cas9 ribonucleoprotein (RNP) complexes targeting both ANP32A and ANP32B genes to obtain double knock-out (dKO) edited clones. Variant ANP32 proteins will be exogenously expressed in dKO cells along with viral polymerase subunits and nucleoproteins corresponding to avian and swine influenza strains. The effect of each variant/isoform on viral polymerase activity will be indirectly assessed by luciferase luminescence using the well-documented Influenza Minigenome Reporter Assay. Additionally, co-immunoprecipitation will be employed to quantify the binding affinity of variant ANP32 proteins and the viral polymerase.