Molecular cloning and transcriptional regulation of Indian peafowl (Pavo cristatus) IFN-α gene

Abstract

Interferon-α (IFN-α) resists viral infections by triggering the transcription of a diverse range of antiviral IFN-stimulated genes (ISGs). However, information about the Indian peafowl (Pavo cristatus) IFN-α (PcIFN-α) has not been reported. In this study, a PcIFN-α gene was amplified, which encoded a protein of 193 amino acids with a 26-amino acid signal peptide sharing 72.16-95.70% identity with other avians in Aves. After expression in prokaryote, PcIFN-α was analyzed for its physicochemical property and antiviral activity. Intriguingly, compared with chicken IFN-α, an effective viral infection therapeutic agent, PcIFN-α showed superior anti-VSV, NDV, and AIV activities, which were then abrogated by rabbit anti-PcIFN-α antibodies in vitro. Moreover, PcIFN-α was shown to be highly sensitive to trypsin; however, it remained stable despite changes in pH and temperature. Additionally, PcIFN-α induced the transcriptional or translational levels of Mx1 and ISG12 genes time-dependently. Overall, the present study revealed that PcIFN-α is a potential novel effective therapeutic agent in antiviral defense responses in peafowl, improving understanding of its involvement in bird antiviral defense.

Keywords: Characterization; Interferon-stimulated genes; Interferon-α; Molecular cloning; Pavo cristatus.

Fig. 1

Cloning, sequence analysis, and 3D prediction of the PcIFN-α. a Amplified PCR product of mature. Lane M, Trans 2 K DNA marker; lane 1, complete sequence of PcIFN-α gene. b The sequence from 1 to 31 AA is the signal sequence. Cysteine residues forming disulfide bonds are marked with gray circles. Letters A–E refer to α-helices in PcIFN-α. c Predicted 3D structures of PcIFN-α. The five α-helices were labeled A–E

Fig. 2

Phylogenetic tree construction of the PcIFN-α and IFN-α AA alignment. a Phylogenetic tree based on combined Neighbour-Joining (NJ), Bayesian Inference (BI) and Maximum Likelihood (ML) methods using a GTR + G + I model for each tree. The NJ/ML bootstrap support is indicated above the tree branch and BI posterior probability support underneath the branch. b IFN-α AA alignment of chicken, Coturnix japonica, Chinese francolin and goose. Identical residues are in black boxes, and similar residues are in grey

Fig. 3

Protein expression and verification of recombinant PcIFN-α. a Lane M: Unstained protein marker; Lane 1: Empty vector pET-33a (+); 2: rHis-PcIFN-α without isopropyl β-D-1-thiogalactopyranoside (IPTG) induction; 3: rHis-PcIFN-α after induction; 4: Sedimentations of rHis-PcIFN-α; 5: Supernatants of rHis-PcIFN-α; b Lane 6: Purified rHis-PcIFN-α; c Lane 7: Western blot analysis of His-tag recombinant PcIFN-α by 6-poly histidine monoclonal antibodies

Fig. 4

Neutralization of PcIFN-α antiviral protective activity. a Negative control (PcIFN-α only); b PcIFN-α and VSV; c PcIFN-α and VSV preincubated with rabbit anti-PcIFN-α antibody; d positive control (VSV only) (TCID₅₀ of VSV titers was 1.048 × 10⁶ U/0.1 ml)

Fig. 5

Effects of PcIFN-α against VSV-, NDV, and AIV-induced cytotoxicity in CEFs. Mean percentage of cell survival in the control group was scaled to 100%. C, control group (CEFs only); IFN, PcIFN-α (PcIFN-α only); Co, co-treatment group (virus and PcIFN-α)

Fig. 6

a pH and b temperature sensitivities of PcIFN-α. PcIFN-α was adjusted to pH 2.0, 4.0, 10.0, and 12.0 at 42, 53, and 65 °C and, then, finally reverted to the original pH (7.0) or cooled down in an ice box. Afterwards, antiviral activities were separately analyzed in F81/VSV system

Fig. 7

Analysis of ISG transcription and Mx-1 protein expression in CEFs after PcIFN-α treatment. a Transcription analysis of ISGs in CEFs after PcIFN-α treatment. b Expression analysis of Mx-1 protein in CEFs after PcIFN-α treatment. CEFs without PcIFN-α treatment were used as control, while GAPDH was used as the internal reference. Data are presented in mean ± SD (*P < 0.05; **P < 0.01)