Differential polymerase activity in avian and mammalian cells determines host range of influenza virus

Abstract

As recently shown, mutations in the polymerase genes causing increased polymerase activity in mammalian cells are responsible for the adaptation of the highly pathogenic avian influenza virus SC35 (H7N7) to mice (G. Gabriel et al., Proc. Natl. Acad. Sci. USA 102:18590-18595, 2005). We have now compared mRNA, cRNA, and viral RNA levels of SC35 and its mouse-adapted variant SC35M in avian and mammalian cells. The increase in levels of transcription and replication of SC35M in mammalian cells was linked to a decrease in avian cells. Thus, the efficiency of the viral polymerase is a determinant of both host specificity and pathogenicity.

FIG. 1.

Transcription and replication activities of SC35 and SC35M in avian and mammalian cells. mRNA, cRNA, and vRNA levels in SC35- and SC35M-infected chicken embryo fibroblasts (CEF) (A), quail fibrosarcoma (QT6) cells (B), human embryo kidney (293T) cells (C), and African green monkey kidney (Vero) cells (D) were determined by primer extension. Primer extension analysis was performed 14 h after inoculation at a multiplicity of infection of 0.1. Transcription products of three independent experiments were quantified using TINA 2.0 software. The results shown are derived from three independent experiments.

FIG. 2.

Transcription and replication activities of SGR and SPM viruses in avian and mammalian cells. Quail fibrosarcoma (QT6) cells (A) and African green monkey kidney (Vero) cells (B) were infected with SC35, SGR viruses (SC35-PB1_SC35M, SC35-PB2_SC35M, SC35-PA_SC35M, and SC35-NP_SC35M), and SPM viruses (SC35-PB2_701N, SC35-PB2_714R, SC35-PB1_13P, and SC35-PB1_678N) at a multiplicity of infection of 0.1. At 14 h postinfection, mRNA, cRNA, and vRNA levels were determined by primer extension. The results shown are derived from three independent experiments.

FIG. 3.

Effects of PB2 mutations D701N and S714R and NP mutation N319K on RNP activities of A/Fowl plague virus (FPV)/Rostock/34 H7N1 in mammalian cells. 293T cells were transfected as indicated with plasmids containing FPV genes PB2, PB1, PA, and NP and FPV mutant genes PB2 D701N, PB2 S714R, and NP N319K. Polymerase activity was determined 24 h after transfection by measuring luciferase activity (7). The baseline value is the result of the cotransfection of plasmids pPol1-NP-Luc and pRSV-lacZ and was subtracted from each measurement. The results shown are derived from four independent experiments.