A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence

Abstract

Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than the parental virus. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern.

FIG 1

The H7N9 NS1 protein is an IFN antagonist in human and chicken cells. Human 293T (A) or chicken DF-1 (B) cells were cotransfected for 16 h with a pCAGGS expression plasmid encoding the indicated NS1 protein (or GST) together with a FF luciferase (FF-Luc) IFN-β promoter reporter plasmid (p125Luc). After infection with a DI-rich SeV preparation for a further 12 h, FF-Luc activity was determined. Results represent the means and standard deviations of triplicate values (normalized to GST + SeV) obtained in a single experiment and are representative of results of two independent experiments. The NS1 sequences (containing silent splice acceptor mutations to prevent NEP/NS2 expression [12]) were derived from A/Texas/36/1991 (Tx/91; human seasonal-like H1N1 virus), A/Wyoming/03/2003 (Wy/03; human seasonal-like H3N2), A/California/04/2009 (Cal/09; human seasonal-like H1N1, previously 2009 pdmH1N1), A/Hong Kong/156/1997 (HK/97; representative of the 1997 H5N1 outbreak), A/Vietnam/1203/2004 (VN/04; representative of the 2004 H5N1 outbreak), A/Shanghai/1/2013 (Sh/1; human H7N9), A/Shanghai/2/2013 (Sh/2; human H7N9), and A/Chicken/Dawang/1/2011 (Dw/11; avian H9N2 with NS1 closely related to H7N9 NS1).

FIG 2

A single I106M substitution in the H7N9 NS1 protein specifically restores efficient CPSF30 binding and inhibition of host cell gene expression. (A) Summary data for a selection of NS1s used in this study. The asterisk denotes predicted binding affinity based on related H3N2 viruses. (B) Binding of NS1 to CPSF30. 293T cell lysate overexpressing FLAG-CPSF30 was mixed with the indicated bacterially expressed 6His-NS1 protein (WT, L103F, I106M, or L103F/I106M [DM] or the 6His multiple cloning site [6His-MCS] negative control) and pulled down (PD) using Ni-nitrilotriacetic acid (NTA) beads. Precipitates eluted after extensive washing were analyzed by SDS-PAGE and Western blotting using anti-NS1 and anti-FLAG antibodies. (C) NS1-mediated inhibition of general host gene expression. Human 293T cells were cotransfected with a pCAGGS expression plasmid encoding the indicated NS1 protein (or GST) together with a constitutively active Renilla-luciferase plasmid. Luciferase activity was determined 24 h posttransfection. Results are given as the means and standard deviations of triplicate values normalized to GST. Statistical significance (**) was determined using the Student t test. (D to F) Binding of NS1 to cellular factors involved in the IFN induction cascade. Experiments were performed as for panel B, using 293T lysates overexpressing FLAG-RIG-I (D), V5-TRIM25 (E), or HA-Riplet (F). Western blotting was performed using appropriate anti-tag antibodies.

FIG 3

Characterization of H7N9-based NS1-WT and NS1-I106M viruses in vitro and in vivo. (A) Virus replication in vitro. Multicycle growth analysis of rSh/1 (6+2) WT and rSh/1 (6+2) NS1-I106M viruses in primary differentiated human airway epithelial (HTBE) cells. Data points show mean values, and error bars represent standard deviations. (B) Induction of IFN-β by the recombinant viruses. MDCK–IFN-β–FF-Luc cells were infected at a multiplicity of infection (MOI) of 2 PFU/cell for 16 h with the indicated virus (or were mock infected) prior to analysis of luciferase activity. Bars represent mean values (n = 3), and error bars represent standard deviations. (C and D) qRT-PCR analyses of viral replication and IFN-β induction in vitro. Primary HTBE cells were infected at an MOI of 2 PFU/cell and lysed at the times indicated. Total RNA was extracted, and following reverse transcription using oligo(dT), the levels of viral M1 mRNA (C) and IFN-β mRNA (D) were quantified in triplicate by qPCR. Values were averaged and normalized to actin mRNA. Mean induction levels relative to mRNA levels in mock-infected cells are shown. Error bars represent standard deviations. Means and standard deviations were calculated from biological triplicates. (E) Virulence in mice. Survival data for 6- to 8-week-old C57/BL6 mice infected intranasally with 10 PFU of the indicated virus (20 mice per virus). Body weights were determined daily for 14 days, and mice showing more than 25% weight loss were considered to have reached the experimental endpoint and were humanely euthanized. (F) Virus replication in vivo. Six- to eight-week-old C57/BL6 mice were infected intranasally with 500 PFU of the indicated virus. Lung titers were determined on days 2 and 4 postinfection from 3 to 4 mice per group. Bars represent mean values. Statistical significance was determined using the Student t test. (G) qRT-PCR analyses of IFN-β induction in vivo. Six-to-eight week-old C57/BL6 mice (3 to 4 mice per group) were infected intranasally with 500 PFU of the indicated virus for 1 or 3 days. Murine IFN-β mRNA was quantified from lung homogenates by qRT-PCR as described for panel D. PBS, phosphate-buffered saline.