Ivermectin Inhibits Bovine Herpesvirus 1 DNA Polymerase Nuclear Import and Interferes with Viral Replication

Abstract

Bovine herpesvirus1 (BoHV-1) is a major bovine pathogen. Despite several vaccines being available to prevent viral infection, outbreaks are frequent and cause important economic consequences worldwide. The development of new antiviral drugs is therefore highly desirable. In this context, viral genome replication represents a potential target for therapeutic intervention. BoHV-1 genome is a dsDNA molecule whose replication takes place in the nuclei of infected cells and is mediated by a viral encoded DNA polymerase holoenzyme. Here, we studied the physical interaction and subcellular localization of BoHV-1 DNA polymerase subunits in cells for the first time. By means of co-immunoprecipitation and confocal laser scanning microscopy (CLSM) experiments, we could show that the processivity factor of the DNA polymerase pUL42 is capable of being autonomously transported into the nucleus, whereas the catalytic subunit pUL30 is not. Accordingly, a putative classic NLS (cNLS) was identified on pUL42 but not on pUL30. Importantly, both proteins could interact in the absence of other viral proteins and their co-expression resulted in accumulation of UL30 to the cell nucleus. Treatment of cells with Ivermectin, an anti-parasitic drug which has been recently identified as an inhibitor of importin α/β-dependent nuclear transport, reduced UL42 nuclear import and specifically reduced BoHV-1 replication in a dose-dependent manner, while virus attachment and entry into cells were not affected. Therefore, this study provides a new option of antiviral therapy for BoHV-1 infection with Ivermectin.

Keywords: BoHV-1; DNA polymerase holoenzyme; Ivermectin; NLS; antiviral; nucleocytoplasmic shuttling; pUL30; pUL42.

Figure 1

Construction and characterization of BoHV-1 UL30-HA and UL42-FLAG expression plasmids. (A) Schematic representation of the BoHV-1 genome (140 kb), along with unique short (US), unique long (UL), terminal repeat (TR), and internal repeat (IR) sequences. Position of UL30 (POL) is located between 45237 nt and 48978 nt on the BoHV-1 genome. (B) UL30 (POL) gene with restriction sites HindIII and EcoRI and the HA tag at C terminus was cloned into the vector pcDNA4. (C) Position of UL42 located between 19596 nt and 20824 nt on the BoHV-1genome. (D) UL42 containing restriction sites HindIII and EcoRI and the FLAG tag at C terminus were cloned into pcDNA4. (E) Restrictive digestion with Hind III and EcoRI. (F) SDS-PAGE/Western blotting assays were performed to detect the indicated fusion proteins. MDBK cells were transfected with indicated plasmids. At 24 h post transfection, the cells were processed for SDS-PAGE/Western blotting assays and the fusion proteins pUL30-HA and pUL42-FLAG were detected with monoclonal antibodies against either HA or FLAG tags. A representative blot of three independent experiments was shown.

Figure 2

Expression of pUL42 is required for nuclear transport of pUL30 in the absence of other viral proteins. MDBK cells were transiently transfected to express the indicated fusion proteins and the cell lysates were subjected to Co-IPs by using anti-HA (A) and anti-FLAG (B) antibodies. Immunoprecipitated proteins were subjected to SDS-PAGE/Western blotting assays to detect the pUL30-HA and pUL42-FLAG fusion proteins using anti-HA (upper panels) or anti-FLAG (lower panels) monoclonal antibodies. The positions of the protein markers (M) are indicated on the left. Data shown are a representative one of three independent experiments. (C) MDBK cells were transiently transfected to express the indicated fusion proteins, processed for in situ immunofluorescence and subjected to CLSM analysis using anti-HA or anti-FLAG monoclonal antibodies. The cell nuclei (DAPI) along with the pUL42-FLAG (FITC) and the pUL30-HA (TRITC) fusion proteins are shown, in grayscale, along with respective RGB merged images of the three channels (merge). RGB profile plots relative to the indicated areas were shown on the right panels (RGB profile). Images are representative of three independent transfection experiments.

Figure 3

IVM impairs nuclear localization of BoHV-1 pUL42.MDBK cells were transiently transfected with pcDNA4-UL42-FLAG in the presence and absence of IVM (25 µM), processed for in situ immunofluorescence and subjected to CLSM analysis at ×63 magnification objective using an anti-FLAG monoclonal antibody. The pUL42 protein is shown in green (FITC); the nucleus is shown in blue (DAPI); and merged images of the two channels are shown on the right (merge).

Figure 4

IVM impairs BoHV-1 replication. (A) MDBK cells were infected with BoHV-1 at MOI of 0.1 PFU/mL and subsequently treated with various concentrations of IVM and DMSO as control. At 24 h post infection, viruses were harvested through three cycles of freezing-thawing, and the virus titers were determined by plaque assay. Data shown are the mean ± SD from three independent experiments. Significance values were calculated using Student’s t test and determined by *p<0.05, **p<0.01, and ***p<0.001 relative to DMSO treated cells. (B) Percentage inhibition of BoHV-1 production after IVM treatment as compared to the control. (C) The results of plaque assay at 10⁻⁴ dilution of IVM treated and untreated cultures.

Figure 5

IVM does not affect cell viability, virus binding and internalization. (A) MDBK cells were incubated with increasing concentrations of Ivermenctin for 48 h. Cell viability was then measured using an MTT assay. Values represent the mean± SD of the three independent experiments. (B) BoHV-1 was pretreated with various concentrations of IVM at 37°C for 1 h and incubated with MDBK cells at a MOI of 0.1 PFU/cell at 4 °C for 1 h. The unbound viruses were removed by washing cells with PBS, and the cells were incubated at 37°C for a further 48 h. (C) MDBK cells were incubated with BoHV-1 at MOI of 0.1 PFU per cell at 4 °C for 1 h; the unbound viruses were removed by washing cells with PBS. Then the cells were treated with increasing concentrations of IVM, and incubated at 37 °C for 30 min. The cells were subsequently washed with citrate buffer (pH 3.0) to remove residual viruses and cultured in maintenance media at 37 °C for 48 hrs. The virus production in (B) and (C) was determined by PRAs. Data shown are the mean ±SD from three independent experiments. Statistical significance was calculated using Student’s t test according to *p<0.05, **p<0.01, and ***p<0.001.