Characterization of chicken IFI35 and its antiviral activity against Newcastle disease virus

Abstract

Interferon-induced protein-35 kDa (IFI35) was an antiviral protein induced by interferon (IFN)-γ, which plays an important role in the IFN-mediated antiviral signaling pathway. Here, we cloned and identified IFI35 in the chicken for the first time. Chicken IFI35 (chIFI35) contains an open reading frame (ORF) of 1,152 bp encoding a protein of 384 amino acids containing two conserved Nmi/IFI35 domain (NID) motifs. Tissue distribution analysis of chIFI35 in healthy and Newcastle disease (ND) virus-infected chickens indicated a positive correlation between chIFI35 mRNA transcription and ND viral loads in various tissues. The role of chIFI35 in regulation NDV replication were further assessed by up- or down-regulated chIFI35 expression in DF-1 cells transfected with plasmid harboring chIFI35, pCMV-3HA-chIFI35 or shRNA targeting chIFI35, pshRNA-chIFI35 plasmids. NDV replications in DF-1 cells were significantly reduced or slightly increased by over- or under-expression of the chIFI35 protein, respectively, indicating the role of chIFI35 in anti-NDV infection. Moreover, chIFI35 also involved in regulation of viral gene transcription and IFNs expression. The collected data were meaningful for research of chicken antiviral immunity and shed light on the pleiotropic antiviral effect of chIFI35 during NDV infection.

Keywords: Newcastle disease virus; antivirus; chicken; interferon; interferon-induced protein-35 kDa.

Fig. 1.

Multiple alignment analysis and phylogenetic tree construction based on the interferon-induced protein-35 kDa (IFI35) amino acid sequences. (A) Multiple alignment analysis of IFI35 family amino acid sequences using Clustal W in DNAstar. These sequences were downloaded from GenBank with accession numbers as follows: XM_418132 (Gallus gallus IFI35, ggaIFI35), XM_010724650 (Meleagris gallopavo IFI35, mgpIFI35), XM_002932512 (Xenopus tropicalis IFI35, xtrIFI35), XM_008329183 (Cynoglossus semilaevis IFI35, fishIFI35), NM_005533 (Homo IFI35, hasIFI35), NM_027320 (Mus musculus IFI35, mmuIFI35) and XM_008113173 (Anolis carolinensis IFI35, acsIFI35). Amino acids conserved among species were indicated as identical “•”. (B) Evolution analyzes were conducted using MEGA6.0. Numbers in branches indicate evolutionary distances. The evolutionary history was inferred using the neighbor-joining (NJ) method. The evolutionary distances were computed using the Poisson correction method.

Fig. 2.

Tissue distribution profile of chicken interferon-induced protein-35 kDa (chIFI35). The relative expression of chIFI35 of F48E9 infected chicken embryo using RNA-Seq and RT-qPCR (A). The relative expression of chIFI35 at 24 hr and 48 hr after F48E9 infected the bursa of fabricius (B). The tissue distribution profiles of chIFI35 in various tissues in healthy chicken (C) and F48E9-infected chickens (D). (E) The viral loads of Newcastle disease virus (NDV) in different tissues.

Fig. 3.

Viral titers of Newcastle disease virus (NDV) in DF-1 cells with interferon-induced protein-35 kDa (IFI35) over- or under-expression. DF-1 cells were transfected with pCMV-3HA-IFI35, empty vector plasmids (A), or interference plasmids (D) for 24 hr, and infected with La Sota (1 moi) or F48E9 (0.1 moi). The viral titer of La Sota (B, E) and F48E9 (C, F) at different time points were detected using the TCID₅₀ method. **P<0.01, *P<0.05, and ns, not significant.

Fig. 4.

Chicken interferon-induced protein-35 kDa (ChIFI35) affects viral gene transcription. (A) The plasmids pCAGGS-HA-P and pCAGGS-Flag-chIFI35 were co-transfected for 36 hr in DF-1 cells, and observed the cell sublocalization of chIFI35 and NDV P protein under the laser confocal microscopy (400×). (B) CT values of each viral gene transcript after F48E9 infection. Data are shown as the mean ± SEM (n=3). The statistical analysis was performed in GraphPad Prism using unpaired two-tailed t-tests: *P<0.05, **P<0.01.

Fig. 5.

Relative expression of chicken interferon-induced protein-35 kDa (chIFI35) and interferons (IFNs) during F48E9 infection. DF-1 cells were infected with F48E9 (0.1 moi, A–D), and the cells were collected at various timepoints to detect the expression changes using RT-qPCR of chIFI35 (A), IFNα (B), IFNβ (C), and IFNγ (D). (E) ChIFI35 was increased by IFNα/β over-expression. (F) IFNα/β/γ expression using RT-qPCR following transfection of DF-1 cells with chIFI35 and empty vector plasmids for 24 hr. Data are shown as the mean ± SEM (n=3). The statistical analysis was performed in GraphPad Prism using unpaired two-tailed t-tests: **P<0.01, ***P<0.001.

Fig. 6.

Positive feedback regulation of interferon (IFN) expression by chicken interferon-induced protein-35 kDa (IFI35). After transfection with chIFI35 in DF-1 or CEF cells for 24 hr, cells were infected with F48E9 for a further 24 hr, and the IFN modulators were detected using RT-qPCR. Data are shown as the mean ± SEM (n=3). The statistical analysis was performed in GraphPad Prism using unpaired two-tailed t-tests: *P<0.05, **P<0.01.