- Short report
- Open Access
Bovine ISG15: an antiviral and inducible protein in BIV infected fetal bovine lung cells
© Liu et al; licensee BioMed Central Ltd. 2010
- Received: 19 March 2010
- Accepted: 23 June 2010
- Published: 23 June 2010
Bovine ISG15 (bISG15) is an interferon inducible ubiquitin-like protein that is responsible for the establishment of early pregnancy in ruminant, understanding the properties of bISG15 capable of being inducible in fetal bovine lung (FBL) cells upon infection of bovine immunodeficiency virus (BIV) is of significant importance. In this study, we investigated the expression of bISG15 in poly I:C treated FBL cells. The increased expression of bISG15 was observed, and the inhibition of BIV replication was also detected in FBL cells. Elimination of bISG15 expression by small interfering RNA reversed the bISG15 mediated inhibition of BIV replication. These findings demonstrate that bISG15 plays an important role in inhibition of the BIV replication in FBL cells. Furthermore, real-time PCR and western blot assay revealed that bISG15's expression can also be induced in BIV infected FBL cells. Taken together, bISG15 is an antiviral and inducible protein in BIV infected FBL cells.
- Luciferase Activity
- Bovine Viral Diarrhea Virus
- Relative Luciferase Activity
- Bovine Immunodeficiency Virus
- Antiviral Function
Type I IFNs' binding to cell type specific receptors can induce the synthesis of a plethora of proteins , many of which play crucial roles in the antiviral response . ISG15 was one of the most strongly induced proteins. Thirty years past since ISG15 was first found , whereas its antiviral function was reported recently . ISG15, a 15-kDa ubiquitin-like protein, can be covalently conjugated to target proteins for post-translational modification through a series of steps similar to ubiquitin modification . ISG15 and its conjugation modification play roles in processes including regulation of IFN signaling, innate immunity, anti-viral infections, pregnancy, and carcinogenesis . Interestingly, ISG15 and enzymes involved in ISG15 modification are interferon inducible [1, 3, 6–8]. This indicates ISG15 and its conjugation play important roles in innate immunity and antiviral response to infection. Mice deficient of ISG15 have been shown to be more susceptible to influenza, herpes, and Sindbis viruses' infection . It has been reported that ISG15 can inhibit the release of human immunodeficiency virus type 1 (HIV-1) virions by disrupting the interaction of the Gag L domain with Tsg101 , and ISG15 can also repress the replication of Ebola virus by inhibiting the release of virions . Unlike ubiquitin or other ubiquitin-like molecules, cross-species' conservation of ISG15 is low . Therefore, it is necessary and intriguing to elucidate the antiviral functions of ISG15 in wide variety of species.
Bovine ISG15 (bISG15) has 70% homology amino acid sequence to human ISG15 . The studies about bISG15 are mainly focused on its role in reproductive biology. The expression of bISG15 is up-regulated at endometrium in response to conceptus-secreted IFN-τ during early pregnancy . The relation between bISG15 and innate immune response has also been noticed. The expression of bISG15 in maternal blood is increased in cows infected with acute non-cytopathic bovine viral diarrhea virus (BVDV) . However, the reports about property of the bISG15 with antiviral function are scarce up to date. Recently, we have found that expression of bISG15 can be induced in fetal bovine lung (FBL) cells after treated with poly I:C or lipopolysaccaride (LPS) . FBL cells were chosen to be as a target type of cells here, since our lab found that FBL cells could be infected by viruses which are capable of infecting cattle, such as BVDV, bovine foamy virus (BFV), and bovine immunodeficiency virus (BIV). BIV can infect FBL cells in vitro, and induce syncytia formation in FBL cells. Moreover, BIV DNA can also be detected in bovine lung tissues . BIV, which has a similar genome to HIV-1, is associated with chronic inflammatory diseases in infected cows . Considering the similarity between BIV and HIV-1, BIV may contribute as alternative animal virus model for certain aspects of HIV research. It has been reported that ISG15 can inhibit the replication of HIV . We previously demonstrated that bISG15 can be induced robustly in poly I:C treated FBL cells . Here, we further try to address whether the replication of BIV can be repressed in FBL cells treated with poly I:C and to address extensively whether the expression of bISG15 is changed in FBL cells infected with BIV.
In this study, FBL cells were isolated from fetal bovine lung issues. The primary FBL cells isolated from the lung issues of fetal calf were called passage"1". When FBL cells proliferated and separated, they were called passage "2" and so on. All the FBL cells used in the experiment were the passages between 10 and 20. BIV indicator cell line, designated as BIVL, has an integrated reporter plasmid which has a firefly luciferase reporter protein gene downstream of BIV LTR promoter . FBL cells and BIVL cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 IU/ml penicillin, 50 μg/ml streptomycin in 5% CO2 at 37°C. Due to fail of obtaining cell free BIV, a BIV strain R29 was used here. The BIV R29 strain was maintained in FBL cells. All the BIV used in the experiment were the FBL cells infected BIV. Poly I:C (purchased from Sigma-Aldrich) was dissolved in PBS at a concentration of 2.5 mg/ml. BIVL cells were plated at a density of 4 × 104 per well in 12-well plates to indicate BIV infection efficiency. BIVL cells co-cultured with BIV infected/uninfected FBL cells at equal cell number. After 12 hours, all cells were assessed by luciferase assays (Promega luciferase assay kit) to indicate the replication efficiency of BIV in FBL cells . Synthetic siRNA of bISG15 (bISG15siRNA) and negative control siRNA (scrambled siRNA) (purchased from GenePharma) were used as annealed oligonucleotide and was resuspended in RNase-free H2O, respectively. The sense sequence of scrambled siRNA is 5'-UUC UCC GAA CGU GUC ACG UTT-3', the anti-sense sequence of scrambled siRNA is 5'-ACG UGA CAC GUU CGG AGA ATT-3'; and the sense sequence of bISG15siRNA is 5'-CAC CGU GUU CAU GAA UCU ATT-3', the anit-sense sequence of bISG15siRNA is 5'-UAG AUU CAU GAA CAC GGU GTT-3'. To validate the suppressing effect of bISG15, bISG15siRNA or scrambled siRNA (100 Nmol/hole) was transfected to FBL cells with Lipofectamine 2000 (Invitrogen), respectively. Realtime PCR and Western blot assay of bISG15 were performed as previously described . Briefly, total RNA from cells was isolated with TRIzol reagent (Invitrogen) following the manufactures's instructions, and then the extracted RNA was reverse transcribed into cDNA by reverse transcriptase (Promega). Real time PCR was performed using an IQ5 Multicolor Real-time PCR detection system (Bio-Rad) and fluorescent EVAGreen nucleic acid stain (Biotium). Primers used to quantify bISG15's cDNA were bISG15-F (5'-GTG GTG CAG AAC TGC ATC TC-3') and bISG15-R (5'-GCC AGA ACT GGT CTG CTT GT-3'). GAPDH cDNA served as endogenous control, GAPDH was amplified using the primers bGAPDH-F (5'-AAC GGC ACA GTC AAG GCA GA-3') and bGAPDH-R (5'-TCG GCA GAA GGT GCA GAG AT-3'). The level of bISG15 transcription was normalized to GAPDH using the 2-ΔΔCt method. The polyclonal murine bISG15 antibody used in western blot assays was obtained in our lab. Western blot detection was performed using the chemiluminescence detection regent (Santa Cruz). To ensure equal protein loading, anti-β-actin antibody (Santa Cruz) was used to detect β-actin expression in cells.
This study demonstrates that bISG15 is an antiviral and inducible protein in BIV infected fetal bovine lung cells. BIV infection can induce the expression of bISG15 in FBL cells, but it is still unclear that the detail involved in this process. It was shown in Figure 3.a, the induction of bISG15 in FBL cells infected with BIV was decreased at 3 hours post-infection, while FBL cells infected with BIV at 18 hours post-infection were still alive, not dead obviously. To infect the naive FBL cells, we added the BIV infected FBL cells to naive FBL cells. All cells involved in this experiment were exclusively FBL cells not other cells. FBL cells infected with BIV may activate some signal pathways like interferon, NF-κB signal pathway and so on. These signal pathways may induce the expression of bISG15 directly or some cytokines such as Type I interferon. These cytokines then induce the expression of bISG15. It indicates that the induction of bISG15's expression in FBL cells by BIV infection may be related to several viral and cellular events via unknown mechanism. Our results suggested that bISG15 played a role in poly I:C-induced inhibition of BIV in FBL cells. The function of bISG15 repressing the replication of BIV in FBL cells demonstrates that bISG15 is a key mediator induced in FBL cells inhibition of BIV replication and also reveals the antiviral function of ISG15 in other species.
The R29 strain of BIV was kindly provided by Dr Charles Wood (University of Nebraska Lincoln). This work was supported by the National Natural Science Foundation of China (30770081) and Tianjin municipal science and technology foundation (08ZCGHHZ01800).
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