Inhibition of infectious pancreatic necrosis virus replication by atlantic salmon Mx1 protein

Abstract

Mx proteins form a family of interferon (IFN)-induced GTPases with potent antiviral activity against various single-stranded RNA viruses in mammals and chickens. In fish, alpha/beta IFN has been reported to inhibit the replication of infectious pancreatic necrosis virus (IPNV), but the mode of action has not been elucidated. A correlation between the inhibition of IPNV and Mx protein expression has, however, been observed. To examine whether Atlantic salmon Mx1 protein (ASMx1) possesses antiviral activity against IPNV, CHSE-214 cells constitutively expressing ASMx1 were established. ASMx1 appeared to be localized in the cytoplasm. The ASMx1-expressing clone selected showed a severely reduced IPNV-induced cytopathic effect, which was confirmed by a 500-fold reduction in virus yield. The antiviral activity against IPNV was further confirmed by the inhibition of virus protein synthesis and the reduced accumulation of virus transcripts. The present work further adds to the body of evidence which suggests that antiviral activity is a major functional role of vertebrate Mx proteins. Moreover, the list of viruses inhibited by Mx proteins is extended to include double-stranded RNA viruses.

FIG. 1.

Analysis of the ASMx1-expressing clone. (a) Mx protein expression was detected by an indirect immunofluorescence assay with a polyclonal antibody generated against rainbow trout Mx3 protein. The cells shown are CHSE-214 cells permanently transfected with ASMx1, CHSE-214 cells incubated with recombinant IFN for 24 h, and CHSE-214 cells permanently transfected with GFP. All cells were monolayers, and the percentage of ASMx1-expressing cells was calculated to be about 65%. (b) Expression of IFN, as determined by RT-PCR, in untreated CHSE-214 cells, the ASMx1-expressing clone, CHSE-214 cells transfected with poly(I-C) for 48 h, and CHSE-214 cells permanently transfected with GFP. Actin bands confirmed the presence of equal amounts of cDNA in all samples. (c) Growth curves for the ASMx1-expressing clone, GFP-expressing CHSE-214 cells, and untreated CHSE-214 cells. Cells seeded in quadruplicate at a confluence of 2 × 10⁴ cells per well in 96-well plates were lysed and counted daily. Bars show standard errors. Passage 2 of the ASMx1-expressing clone was used in all experiments.

FIG. 2.

Antiviral activity of the ASMx1-expressing clone against IPNV. The ASMx1-expressing clone (passage 2), CHSE-214 cells treated with recombinant IFN for 24 h, CHSE-214 cells permanently transfected with GFP, and untreated CHSE-214 cells were seeded in 96-well plates and infected with IPNV (MOI, 0.1). (a) For the CPE reduction assay, the cells were fixed and stained with crystal violet in ethanol at 72 h postinfection, and the OD₅₅₀ was measured. Results are presented as the percentage of surviving cells, calculated as the OD₅₅₀ of infected cells (n = 12) divided by the OD₅₅₀ of uninfected cells (n = 4). Bars show standard deviations. (b) The virus yield of the supernatants at 72 h postinfection was determined by the TCID₅₀ method with CHSE-214 cells.

FIG. 3.

Reduction in antiviral activity and progressive loss of Mx protein expression in the ASMx1-expressing clone during selective growth. (a) For each passage (2 to 6), the ASMx1-expressing clone was infected with IPNV (MOI, 0.1). Untreated CHSE-214 cells were included as a control. For the CPE reduction assay, cells were fixed and stained with crystal violet in ethanol at 72 h postinfection, and the OD₅₅₀ was measured. Results are presented as the percentage of surviving cells, calculated as the OD₅₅₀ of infected cells (n = 12) divided by the OD₅₅₀ of uninfected cells (n = 4). Bars show standard deviations. (b) The virus yield of the supernatants from infected cells was determined by the TCID₅₀ method with CHSE-214 cells. (c) Cell extracts from the ASMx1-expressing clone grown in 24-well culture plates for 3 days were harvested for each passage (2 to 6) and analyzed for Mx protein and actin expression by Western blotting. Histograms express the density values of Mx protein bands normalized to the density values of the corresponding actin bands.

FIG. 4.

Inhibition of IPNV protein synthesis in the ASMx1-expressing clone. The ASMx1-expressing clone (passage 2), CHSE-214 cells treated with recombinant IFN for 24 h, CHSE-214 cells permanently transfected with GFP, and untreated CHSE-214 cells were seeded in 24 wells and infected with IPNV (MOI, 0.1). Cell extracts were harvested at 48 h postinfection and analyzed for the expression of IPNV proteins (VP1, VP2, VP3, and nonstructural protein [NS]) by immunoblotting. To show the expression of Mx protein and actin, the blot was stripped and incubated with anti-Mx protein antibody and with antiactin antibody, respectively. The experiment was done twice.

FIG. 5.

Decrease in IPNV transcription in the ASMx1-expressing clone. Untreated CHSE-214 cells, the ASMx1-expressing clone (passage 2), CHSE-214 cells transfected with poly(I-C) for 24 h, and CHSE-214 cells permanently transfected with GFP, all grown as monolayers in six-well plates, were infected with IPNV (MOI, 0.1). Total RNA was harvested at 24 h postinfection, and the expression of VP2 transcripts of IPNV and Mx protein transcripts was analyzed by semiquantitative RT-PCR. The amount of cDNA in each sample was verified by actin-specific RT-PCR.