Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase.

Yukari Miyake,Ayae Honda,Kosuke Ishii

doi:10.3389/fmicb.2017.00162

Abstract

Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase.

Highlights

The genome of influenza virus is composed of eight negative RNA molecule segments (Palese and Shaw, 2007)
To understand the mechanism underlying the conversion of influenza virus RNA-dependent RNA polymerase (RdRp) function in host cells, we carried out yeast two-hybrid screening to identify host factor(s) interacting with RdRp and which RdRp subunit(s) were involved in the interaction using recombinant plasmids (Honda et al, 2007), as described in Section “Yeast Two-Hybrid Screening.”
The RdRp enzyme can catalyze the synthesis of three species of RNA (Krug et al, 1989); purified RdRp from recombinant baculovirus coinfected with Tn5 requires a primer for RNA synthesis, and cannot synthesize all three different RNAs

Summary

Introduction

The genome of influenza virus is composed of eight negative RNA molecule segments (Palese and Shaw, 2007). The influenza virus invades the cytoplasm of host cells in endosomes by Influenza RdRp Interacts with PKM binding to sialic acid on cellular membranes via hemagglutinin (HA), a viral glycoprotein on the envelope of the influenza viral particle (Lakadamyali et al, 2004), and is transported to the vicinity of the nucleus, where decapsidation of the virus occurs and a vRNP complex, consisting of viral RNA (vRNA), RdRp, and NP is released from the endosome (Lakadamyali et al, 2004). RdRp catalyzes the synthesis of three species of RNA (Krug et al, 1989): complementary RNA (cRNA), messenger RNA (mRNA), and vRNA. Initiation of mRNA synthesis requires host capped RNA as a primer, while neither vRNA nor cRNA synthesis rely on a primer for initiation. RdRp synthesizes mRNA but not cRNA or vRNA (Honda et al, 1990); while in virusinfected cells, the same enzyme synthesizes mRNA, and cRNA and vRNA

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Results

Conclusion