Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production.

Patrick D Slaine,Mariel Kleer,Cara Macrae,Michelle Warhuus,Todd Hatchette,Denys A Khaperskyy,Sarah Mcalpine,Emily Lamoureux,André M Comeau,Craig Mccormick

doi:10.3390/v10050272

Abstract

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

Highlights

Influenza A viruses (IAVs) evolve rapidly and exist as genetically heterogeneous populations known as quasispecies
Madin-Darby canine kidney (MDCK) cells, human embryonic kidney 293T (HEK293T) cells and mouse L cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA)
At three days post infection, mice were euthanized, and lung tissue was homogenized in phosphate buffered saline (PBS) to release infectious virions, which were used to infect the cohort of mice

Summary

Introduction

Influenza A viruses (IAVs) evolve rapidly and exist as genetically heterogeneous populations known as quasispecies. Viruses 2018, 10, 272 co-infection of host cells with two or more genetically-distinct viruses can result in re-assortment of genome segments into hybrid progeny viruses with new properties. This genetic re-assortment is known as antigenic shift [1]. IAV encodes an error-prone RNA-dependent RNA polymerase (RdRp) that misincorporates 2–3 ribonucleotides into each newly-synthesized genome, in a process known as antigenic drift [2]. These processes accelerate viral evolution and allow beneficial mutations to be fixed in the viral genome. Antigenic shift and antigenic drift increase the plasticity of the IAV genome, which enables rapid emergence of viral progeny with new properties [6]

Methods

Results

Conclusion