PB2-588I enhances 2009 H1N1 pandemic influenza virus virulence by increasing viral replication and exacerbating PB2 inhibition of beta interferon expression

Abstract

The 2009 pandemic H1N1 influenza virus (pdm/09) is typically mildly virulent in mice. In a previous study, we identified four novel swine isolates of pdm/09 viruses that exhibited high lethality in mice. Comparing the consensus sequences of the PB2 subunit of human isolates of pdm/09 viruses with those of the four swine isolate viruses revealed one consensus mutation: T588I. In this study, we determined that 588T is an amino acid mutation conserved in pdm/09 viruses that was exceedingly rare in previous human influenza isolates. To investigate whether the PB2 with the T5581 mutation (PB2-T558I) has an effect on the increased pathogenicity, we rescued a variant containing PB2-588I (Mex_PB2-588I) in the pdm/09 virus, A/Mexico/4486/2009(H1N1), referred to as Mex_WT (where WT is wild type), and characterized the variant in vitro and in vivo. The results indicated that the mutation significantly enhanced polymerase activity in mammalian cells, and the variant exhibited increased growth properties and induced significant weight loss in a mouse model compared to the wild type. We determined that the mutation exacerbated PB2 inhibition of mitochondrial antiviral signaling protein (MAVS)-mediated beta interferon (IFN-β) expression, and PB2-588I was observed to bind to MAVS more efficiently than PB2-588T. The variant induced lower levels of host IFN-β expression than the WT strain during infection. These findings indicate that the pdm/09 influenza virus has increased pathogenicity upon the acquisition of the PB2-T588I mutation and highlight the need for the continued surveillance of the genetic variation of molecular markers in influenza viruses because of their potential effects on pathogenicity and threats to human health.

FIG 1

Viral RNA polymerase activity. Polymerase activity assays were performed in human 293T cells at 33°C and 37°C by transfecting vectors expressing the polymerase subunits and a pNP-Luc luciferase reporter. After cells were cultured at 33°C and 37°C for 24 h, luciferase production was measured. The data were normalized to the activity observed for the polymerase subunits and are presented on a percentage scale; the results are presented as the means ± standard deviations of three independent experiments. **, P < 0.001, as determined by a t test.

FIG 2

Growth kinetics of recombinant viruses in cells. A549, MDCK, and PK-15 cells were infected with the WT or variant viruses at an MOI of 0.001. At the indicated hours postinfection (hpi), the virus titers in the supernatant were determined with the MDCK cells. The reported values are presented as the means ± standard deviations of three independent experiments. **, P < 0.001, as determined by a t test.

FIG 3

Pathogenicity of the WT and the variant in mice. Six mice per group were intranasally inoculated with 10⁴ TCID₅₀s/ml, 10⁵ TCID₅₀s/ml, and 10⁶ TCID₅₀s/ml (each in 50 μl) of the WT or the variant. (A) Mouse body weights were monitored daily for 14 days. The values represent the average scores of the overall body weight loss compared with the initial body weight ± standard deviations. (B) The percent survival values were calculated by observing the infected mice. (C) The mice were intranasally inoculated with 10⁵ TCID₅₀s/ml of the WT or the variant virus. Tissues were collected from the mice (n = 3) on the indicated days postinfection, and the virus titers were determined in MDCK cells. *, P < 0.01, **, P < 0.01, as determined by a t test.

FIG 4

Histopathology analyses. The lungs of the infected mice were fixed with formalin, embedded in paraffin, and stained with hematoxylin and eosin. The images were obtained at a magnification of ×20. Arrow a, infiltration of inflammatory cells; arrow b, alveolar wall thickening; arrow c, alveolar wall capillary hyperemia; arrow d, focal lymphocyte infiltration.

FIG 5

PB2-588I exacerbates the inhibition of IFN-β. (A to D) The transfection of 293T cells was performed with a luciferase reporter plasmid under the control of an IFN-β promoter and a Renilla control plasmid, PB2 expression plasmids, and plasmids expressing either RIG-I, MDA-5, MAVS, or TBK-1. After 24 h, the cells were lysed, and the luciferase and Renilla activities were measured. The Renilla-adjusted luciferase activity (RLU) in the presence of overexpressed RIG-I, MDA-5, MAVS, or TBK-1 but in the absence of the PB2 protein (empty vector) was set to 100%. The activity in the presence of the PB2 protein was expressed as a percentage of that of the empty vector control. (E and F) The transfection of the 293T cells was performed with a luciferase reporter plasmid under the control of an NF-κB or IRF-3 promoter, a MAVS expression plasmid, and a plasmid expressing the PB2 protein or an empty vector. (G) The transfection of the 293T cells was performed with a luciferase reporter plasmid under the control of an IFN-β promoter, a MAVS expression plasmid, and a plasmid expressing either the wild-type PB2 or the PB2-T588I mutant protein. (H) The lysates from the 293T cells transfected with Flag-MAVS and PCMV-Myc-PB2-588T or PCMV-Myc-PB2-588I and immunoprecipitates were analyzed by Western blotting using anti-Flag and anti-Myc antibodies. (I) Densitometry analysis of the Western blot results of the binding efficiency of coimmunoprecipitate PB2-588I and PB2-588T with Flag-MAVS was done using ImageJ (NIH); the value of PB2-588T was set as the standard, and values shown are ratios of their bands to the standard. The bars represent the standard errors of the means, based on three experiments. *, P < 0.01; **, P < 0.001 (as determined by a two-tailed Student's t test). IP, immunoprecipitation; a-, anti.

FIG 6

The Mex_PB2-588I induced lower levels of IFN-β expression than the Mex_WT virus. (A) A549 cells were infected with either the Mex_WT or the Mex_PB2-588I virus at an MOI of 0.1 TCID₅₀/ml. At 12 and 24 h postinfection, total RNA was isolated, and RT-qPCR was performed to measure IFN-β mRNA levels. (B) Twelve mice per group were infected with either the Mex_WT or the Mex_PB2-588I virus at 10⁵ TCID₅₀s/ml. At 1, 3, 5, and 7 dpi, IFN-β was measured using a mouse IFN-β ELISA kit. Columns represent mean observed concentrations of cytokines from three mice. The error bars represent the standard errors of the means. *, P < 0.01; **, P < 0.001 (as determined by a two-tailed Student's t test).