Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity

Abstract

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 μm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Figure 1

(a) Schematic representation of recombinant IHNV genomic RNAs indicating gene order and expressed proteins. G_U and G_M refer to the glycoprotein genes from the IHNV U‐type and M‐type strains, respectively. NV_U and NV_M refer to the non‐virion genes from the IHNV U‐type and M‐type strains, respectively. (b) Comparative growth of IHNVs and rIHNVs in RTG‐2 cells. The cells were infected at a multiplicity of infection of 1 PFU per cell, and samples of the supernatant medium were collected at the indicated time points. The samples were titrated in duplicate by plaque assay on EPC cells. The results are presented as the means ± SD of three independent experiments. IHNV, infectious haematopoietic necrosis virus; EPC, epithelioma papulosum cyprini.

Figure 2

(a) Effect of poly I:C pretreatment on the growth of U‐ and M‐type IHNV in RTG‐2 cells. Cells were pre‐incubated with poly I:C at 25 μg mL⁻¹ for 24 h. Then, cells were infected with IHNV U‐ or M‐type virus at a multiplicity of infection (MOI) of 1 PFU per cell, and samples of the supernatant medium were collected at the indicated time points. The samples were titrated in duplicate by plaque assay on EPC cells. The results are presented as the means ± SD of three independent experiments; ns, not significant, *P < 0.05, ***P < 0.05 vs. virus titre at 0 h. (b) Effect of poly I:C treatment after virus infection. RTG‐2 cells were infected with IHNV U‐ or M‐type strains at an MOI of 1 PFU per cell for 24 h prior to poly I:C treatment. After further incubation for 24 h, samples of the supernatant medium were collected and titrated in duplicate by plaque assay on EPC cells. The results are presented as the means ± SD of three independent experiments. IHNV, infectious haematopoietic necrosis virus; EPC, epithelioma papulosum cyprini.

Figure 3

Analysis of interferon (IFN)‐related responses in RTG‐2 cells infected with U‐type and M‐type IHNV. (a) IFN1 and Mx1 expression in IHNV‐infected RTG‐2 cells. RTG‐2 cells were harvested at indicated times after U‐ or M‐type IHNV infection, or mock infection, and total RNA was analysed with real‐time PCR for IFN1 and Mx1. The expression level of IFN1 and Mx1 in mock‐infected cells at 0 h was defined as one. The results are presented as the means ± SD of three independent experiments. (b) Assay for functional IFN1 by luciferase activity in RTG‐P1 cells after incubation for 24 h with supernatants from mock‐infected or virus‐infected fish cells (RTG‐2 or EPC cell lines). The positive control is RTG‐2 cells stimulated with 25 μg mL⁻¹ poly I:C. The results are presented as the means ± SD of three independent experiments. (c) The effect of PKR inhibitor on the growth of U‐ and M‐type IHNV. RTG‐2 cells were infected with IHNV U‐ or M‐type strains at a multiplicity of infection of 1 PFU per cell in the presence or absence of the PKR inhibitor, 2‐AP (1.5 mm). The samples of the supernatant medium were collected at the indicated time points and titrated in duplicate by plaque assay on EPC cells. The results are presented as the means ± SD of three independent experiments, *P < 0.05. IHNV, infectious haematopoietic necrosis virus; EPC, epithelioma papulosum cyprini.

Figure 4

(a) Schematic representation of P genes showing five potential predicted phosphorylation sites, which are different between U‐ and M‐type viruses. (b,c,d) Effects of inhibitors against the three host kinases PKC, p38MAPK, and casein kinase II (CKII) on the growth of U‐ and M‐type IHNV strains. RTG‐2 cells were pretreated with (b) PKC inhibitor (bisindolylmaleimide XI hydrochloride), (c) p38MAPK inhibitor (SB203580) or (d) CKII inhibitor (5,6‐dichlorobenzimidazole riboside DRB) at the concentrations indicated for 1 h and then infected with IHNV at a multiplicity of infection of 1. The samples of the supernatant medium were collected at the indicated time points and titrated in duplicate by plaque assay on EPC cells. The results are presented as the means ± SD of three independent experiments, **P < 0.005. IHNV, infectious haematopoietic necrosis virus; EPC, epithelioma papulosum cyprini.