Beta interferons from the extant camelids: Unique among eutherian mammals

Abstract

The COVID-19 pandemic is a wake-up call on the zoonotic viral spillover events and the need to be prepared for future outbreaks. Zoonotic RNA viruses like the Middle East respiratory syndrome coronavirus (MERS-CoV) are potential pathogens that could trigger the next pandemic. Dromedary camels are the only known animal source of MERS-CoV zoonotic infections, but little is known about the molecular antiviral response in this species. IFN-β and other type-I interferons provide the first line of defense against invading pathogens in the host immune response. We identified the IFNB gene of the dromedary camel and all extant members of the family Camelidae. Camelid IFN-β is unique with an even number of cysteines in the mature protein compared to other eutherian mammals with an odd number of cysteines. The viral mimetic poly(I:C) strongly induced IFN-β expression in camel kidney cells. Induction of IFN-β expression upon infection with camelpox virus was late and subdued when compared to poly(I:C) treatment. Prokaryotically expressed recombinant dromedary IFN-β induced expression of IFN-responsive genes in camel kidney cells. Further, recombinant IFN-β conferred antiviral resistance to camel kidney cells against the cytopathic effects of the camelpox virus, an endemic zoonotic pathogen. IFN-β from this unique group of mammals will offer insights into antiviral immune mechanisms and aid in the development of specific antivirals against pathogens that have the potential to be the next zoonotic pandemic.

Keywords: Antiviral; Coronavirus; Dromedary camel; Interferon; Poxvirus; Zoonosis.

Fig. 1

Nucleotide sequence of the cloned dromedary camel IFN-β cDNA sequence: We amplified a 646 bp fragment with CdIFNbNF and NR primers. This fragment contained a 561 bp ORF (1–561) encoding a 186 amino acid protein. The start and the stop codons of the ORF are highlighted and the translation of the amino acid is provided below the nucleotide sequence. Later the 5′ and 3′ UTR regions were identified by Rapid amplification of cDNA ends (RACE). The complete IFN-β cDNA sequence (NCBI GenBank Accession No ON256405) consists of 88 bp 5′UTR, 561 bp ORF, and 231 bp 3′UTR with poly-A tail. Analysis of the predicted protein indicated that the first 21 amino acids (underlined) formed the eukaryotic signal peptide. The four conserved cysteine residues in the mature protein are also marked in yellow highlight. The predicted polyadenylation signal sequence (AATAAA) sequence located 187 bp downstream of the stop codon is underlined. The nonamer AU-rich sequence motif TTATTTATT located 156 bp after stop codon that mediates ARE-dependent mRNA decay is also marked in underline.

Fig. 2

Sequence of the cloned 5′ region of the Camelus dromedarius IFNB gene containing the regulatory/promoter elements. Four PRD domains of the Virus Responsive Elements that are reported to regulate the IFNB promoter are marked. The TATA box is also marked in bold. The start codon is highlighted in green.

Fig. 3

Comparison of the IFN-β protein sequence of the dromedary and other camelids to their mammalian orthologs. Downward arrow indicates the predicted signal peptide cleavage site. The conserved cysteine residues in the mature protein are marked in yellow highlight. A single and a double underline respectively indicate potential binding sites for IFNAR1 and IFNAR2. The locations of the predicted helices predicted with the I-TASSER program are marked.

Fig. 4

A phylogenetic analysis of camelid IFN-β proteins and their mammalian orthologs. The evolutionary history was inferred using the Neighbor-Joining method using ClustalW multiple alignment of the IFN-β amino acid sequences. Scale for given branch length indicates 0.1 amino acid substitutions per site. The numbers enclosed in circles indicate the number of cysteines in each organism's mature IFN-β protein.

Fig. 5

- Expression of IFN-β mRNA in camel kidney cells after poly(I:C) treatment or camelpox virus (CMLV) infection: A) Poly(I:C) transfection enhances IFN-β mRNA expression in camel kidney cells. Poly(I:C) (5 μg/mL) was added to the medium or transfected into cells using FuGENE HD transfection reagent, and the cells were grown for 24 h. The fold expression of the IFN-β was estimated with that of the corresponding medium control using GAPDH mRNA as the housekeeping gene. B) Poly(I:C) transfected cells (FuGENE HD, 5 μg/mL) were incubated for 0, 2, 4, 6, 12 & 24 h and the fold expression of IFN-β estimated. C) mRNA expression of IFN-β mRNA in camel kidney cells infected with camelpox virus in vitro: Camel kidney cells were cultured in vitro and infected with CMLV. The cells were incubated for the indicated time (2–24 h) and the fold expression of IFN-β was calculated. The results are shown as mean ± SD (n = 3). Data was analyzed for statistical significance by one-way ANOVA using the Tukey's (A) and Games-Howell (B and C) post-hoc tests. **P-value < 0.01, ***P-value < 0.001 and ****P-value < 0.0001.

Fig. 6

Expression of interferon-stimulated genes (ISG) in dromedary camel kidney cells in vitro after interferon treatment: Camel kidney cells were treated with 100 ng/mL of recombinant camel IFN-β, IFN-α1, a His-tagged control protein, or phosphate-buffered saline (PBS) for 4 h. After treatment, total RNA was isolated and mRNA expression of ISGs (ISG15, Viperin (VIP), Mx1, OAS1, RIG1, and IRF1) was determined by RT-qPCR. The fold induction of the ISGs was estimated with that of the corresponding untreated control using GAPDH mRNA as the housekeeping gene. The data is represented as the mean ± standard deviation (n = 3). Statistical significance (one-way ANOVA with Games-Howell's multiple comparisons test) is indicated for pairwise comparisons compared to the untreated control: *P-value < 0.05, **P-value < 0.01, ***P-value < 0.0001 and ns for no significance. Capped lines connecting two data bars indicate significance between the IFN-β, and IFN-α1 treatments.

Fig. 7

- Antiviral activity of recombinant camel IFN-β: A) Camel kidney cells were pre-treated with medium (Untreated control), His-tagged control protein, recombinant dromedary IFN-α1, or recombinant dromedary IFN-β for 1h and then infected with CMLV (MOI 0.1). The cells were grown for a further 24h and observed, and images were captured with the NIS-Elements D 3.10 software connected to Nikon Eclipse Ti–S inverted microscope. B) Reduction in CMLV DNA after infection of IFN treated cells with CMLV:. The culture lysate was collected 24h post infection and viral DNA isolated. CMLV DNA was estimated by TaqMan qPCR assay using primers CLMV-qTF/CLMV-qTR and 5′-FAM-labeled CMLV QT Probe which recognized the CMP48L gene of CMLV. A standard curve made from 10-fold serial dilutions of viral DNA standard run under the same conditions in qPCR was used to estimate the viral DNA quantity. The results are shown as mean ± SD (n = 3). Statistical significance (one-way ANOVA with Games-Howell's multiple comparisons test) annotations above the data bars indicate significance values relative to the Untreated Control. Statistical significances between other groups are indicated by lines. ***P < 0.001.