Sendai Virus, a Strong Inducer of Anti-Lentiviral State in Ovine Cells

Abstract

Small ruminant lentiviruses (SRLVs) are widely spread in the ovine and caprine populations, causing an incurable disease affecting animal health and production. Vaccine development is hindered owing to the high genetic heterogeneity of lentiviruses and the selection of T-cell and antibody escape mutants, requiring antigen delivery optimization. Sendai virus (SeV) is a respiratory paramyxovirus in mice that has been recognized as a potent inducer of innate immune responses in several species, including mouse and human. The aim of this study was to stimulate an innate antiviral response in ovine cells and evaluate the potential inhibitory effect upon small ruminant lentivirus (SRLV) infections. Ovine alveolar macrophages (AMs), blood-derived macrophages (BDMs), and skin fibroblasts (OSFs) were stimulated through infection with SeV encoding green fluorescent protein (GFP). SeV efficiently infected ovine cells, inducing an antiviral state in AM from SRLV naturally-infected animals, as well as in in vitro SRLV-infected BDM and OSF from non-infected animals. Supernatants from SeV-infected AM induced an antiviral state when transferred to fresh cells challenged with SRLV. Similar to SRLV, infectivity of an HIV-1-GFP lentiviral vector was also restricted in ovine cells infected with SeV. In myeloid cells, an M1-like proinflammatory polarization was observed together with an APOBEC3Z1 induction, among other lentiviral restriction factors. Our observations may boost new approximations in ameliorating the SRLV burden by stimulation of the innate immune response using SeV-based vaccine vectors.

Keywords: APOBEC3; Sendai virus; innate immunity; interferon; small ruminant lentivirus.

Figure 1

Sendai virus (SeV)-green fluorescent protein (GFP) infection of ovine cells. Fluorescence microscopy images of alveolar macrophages (AMs) (A), blood derived macrophages (BDMs) (B), and ovine skin fibroblasts (OSFs) (C) infected with Sendai virus vector expressing the GFP (right panel) at a multiplicity of infection (MOI) of 10. Bright field images are shown in the left panel. The three cell types and all cells in the three cultures are GFP-positive. Ovine fibroblasts remained GFP-positive after 13 in vitro culture passages ((C), third image).

Figure 2

Differentiation markers in ovine myeloid cells infected with Sendai virus (SeV). Relative expression of M1 (A3Z1, TNF-α) and M2 (MR, DC-SIGN) differentiation markers measured in alveolar macrophages (A) and blood derived macrophages (B). Values are the median (±interquartile range) of at least three independent experiments. * p < 0.05 (paired Mann–Whitney U Test).

Figure 3

Small ruminant lentivirus (SRLV) replication in ovine cells in the context of SeV. SRLV restriction in ovine alveolar macrophages from chronically infected animals (A), or non-SRLV infected animals’ blood derived macrophages (B) and skin fibroblasts (C) that were mock, or Sendai virus (SeV) infected and challenged later on with SRLV. SRLV viral DNA (left panel), Gag-p25 mRNA relative expression (mid panel), and retrotranscriptase (RT) activity (right panel) was measured in AMs of infected animals or BDMs or OSFs from uninfected animals infected with SeV at an MOI of 10 (grey bars). BDMs and OSFs were further infected with SRLV at an MOI of 0.5 (white bars). Viral DNA was measured at 16 h post-infection, p25 mRNA was measured at 48 h post-infection, and RT activity was measured by SYBR Green based real time PCR enhanced reverse transcriptase assay (SG-PERT) in clarified supernatants at 72 h post-infection. Data shown are the median (±interquartile range) and differences were analyzed using the Wilcoxon paired test (* p < 0.05, ** p < 0.01).

Figure 4

Pseudotyped Human Immunodeficiency virus (HIV-1) restriction after Sendai virus (SeV) infection. ((A), left panel) HIV-1-GFP proviral load in ovine skin fibroblasts (OSFs; left axis) and blood-derived macrophages (BDMs; right axis) infected with HIV-1 GFP-based vector or SeV-infected. Values represent the geometric mean copy values (±95% confidence interval (CI)) per 100 ng of total DNA. ((A), right panel) GFP proviral copies measured in uninfected and SeV-infected OSF and BDM transduced with HIV-1 GFP vector. Values are the geometric mean copies (±95% CI) per 100 ng of total DNA. Differences were statistically analyzed using unpaired T test (one-tailed), * p < 0.05. (B) Relative infectivity in 293-T cells of HIV-1-GFP pseudovirus produced in uninfected 293-T cells (control; white bars) or infected with SeV (SeV; grey bars) in fresh 293-T cells. Values are the geometric mean (±95% CI) of at least three independent experiments. Differences were statistically analyzed using paired T test (one-tailed), * p < 0.05, ** p < 0.01.

Figure 5

Lentiviral restriction factors mRNA expression in ovine cells after infection with Sendai virus (SeV). Ovine APOBEC3 proteins (A3Z1 and A3Z2Z3), tetherin (OBST2), TRIM5α, SAMHD1, and RIG-I mRNA expression was quantified in control (white) and SeV-infected (grey) ovine alveolar macrophages (A), blood derived macrophages (B), and skin fibroblasts (C). Values are the median (±interquartile range) of at least three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 (paired Mann-Whitney U test).

Figure 6

Antiviral activity induction after infection with Sendai virus (SeV). (A) RT activity in ovine skin fibroblasts (OSFs) cultured with supernatants from alveolar macrophages (AMs), infected or not with SeV, were infected with SRLV after 24 h of supernatant treatment. SRLV virus production was measured as retrotranscriptase (RT) activity in the supernatant at 72 h post infection. Data shown are the geometric mean ±95% CI of at least three independent experiments. Differences were statistically analyzed using unpaired T test, * p < 0.05. (B) Relative mRNA expression of the restriction factors after infection with SRLV. Data shown are the mean ± SEM of at least three independent experiments. * p < 0.05 (paired Mann–Whitney U test). (C) Relative mRNA expression of restriction factors: ovine APOBEC3Z1 (A3Z1) tetherin (oBST2), TRIM5α, and SAMHD1 measured by quantitative RT-PCR in ovine skin fibroblast (OSF) cultured with supernatants from AM control or AM infected with SeV. Values are the median (±interquartile range) of at least three independent experiments. * p < 0.05 (Mann–Whitney paired U Test). (D) Type-I interferon (IFN) quantification measured by an ovine-adapted IFN bioassay using supernatants from AM control or infected with SeV. Data shown are the median ±interquartile range of at least three independent experiments. * p < 0.05, ** p < 0.01 (Mann–Whitney paired U test).