Subcellular localization of viral proteins in PK15 cells coinfected with PCV2 and CSFV.

<p>PK15-CSFV cells were seeded on glass bottom dishes and inoculated with PCV2 at MOI = 10. At indicated time points after PCV2 infection, cells were fixed and immunostained for PCV2 Cap (green) and CSFV E2 protein (red) as well as DAPI-stained for the nucleus (blue) and observed using CLSM and super-resolution microscopy. The red bar in the merged image represents 10 μm. (A) Overview of PCV2 infection at 72 hpi in PK15-CSFV and ST-CSFV cells. White arrows indicate double-stained cells. (B) Dynamic subcellular localization of viral proteins of PCV2 and CSFV in PK15-CSFV cells. (C) Super-resolution microscopy of PCV2 and CSFV in cells. Cells were fixed and immunostained for N-STORM. Images were taken and reconstructed to obtain 3D models of colocalization of PCV2 Cap and CSFV E2 proteins in the nucleus (white arrows) and in the cytoplasm (blue arrows).</p

PCV2-induced apoptosis.

<p>(A) Cells infected with PCV2 (MOI = 1) were fixed for the TUNEL assay at 72 hpi and immunostained for viral proteins. Apoptotic cells (green), PCV2 Cap proteins (red), CSFV E2 proteins (blue) and nuclei (grey) are shown. (B) Statistical analysis of PCV2-induced apoptosis. Total numbers of TUNEL-positive, PCV2-positive and TUNEL-PCV2 dual-positive cells were counted, and proportions of each type of cells in all labeled (TUNEL or immunostained) cells were calculated in four random fields. (C and D) Cells inoculated with PCV2 of different MOIs were determined by TUNEL assay (C) and measurement of luminescent caspase–3/7 activities (D). The red bar in the merged image indicates 10 μm. Data are represented as means ± SD (n = 3 or more; ns, <i>P</i> > 0.05).</p

Viral infection status of CSFV in PK15 cell line harboring replicating CSFV.

<p>PK15 cells were infected with CSFV and then serially subcultured. Different passages of PK15-CSFV harboring replicating CSFV were collected to detect the viral infection status. (A) Histogram and proportion of CSFV-positive cells by flow cytometry. The red line indicates the positive gate for the cell population. (B) Three indexes of viral infection of PK15-CSFV cells: proportion of CSFV-positive cells using flow cytometry, viral genomic copies in virus stocks by absolute quantitative real-time PCR and infectivity of virus stocks by measurement of TCID₅₀. Data are represented as means ± SD (n = 3).</p

Replication of PCV2 and CSFV in PK15-CSFV cells.

<p>PK15 and PK15-CSFV cells were inoculated with PCV2 at MOI = 0.5, 1, 4, 7, 10 and 15. At 72 hpi, cells and supernatants were freeze-thawed to obtain virus stocks. Titers were determined by measurement of TCID₅₀ in PK15 or ST cells and by absolute quantitative real-time PCR. (A) TCID₅₀ of PCV2. (B) Genomic copies of PCV2. (C) TCID₅₀ of CSFV. (D) Genomic copies of CSFV. (E) Total RNA of cells was extracted, and relative quantitative real-time PCR was used to detect the replication level of CSFV in PK15-CSFV cells infected with PCV2 at different MOIs. The ratio of CSFV mRNA to β-actin mRNA in mock-infected PK15-CSFV was defined as 1, and then the relative CSFV mRNA ratio in PCV2-infected PK15-CSFV cells was determined. Data are represented as means ± SD (n = 3; ns, <i>P</i> > 0.05; *<i>P</i> < 0.05; **<i>P</i> < 0.01).</p

Infectivity of PCV2 and CSFV in different porcine cell lines.

<p>3D4/31, ST and PK15 cell lines were inoculated with PCV2 at MOI = 1, 4, 7 and 10 or with CSFV at MOI = 0.03 and 0.1, respectively. After culture for 72 h, cells were fixed and stained for IFA, and then four fields were randomly chosen to count the percentage of the positive cells. Meanwhile, the cells were freeze-thawed to obtain virus stocks for determining virus titers. The percentage of PCV2-postive cells (A), PCV2 titer (B), percentage of CSFV-positive cells (C) and CSFV titer (D) were determined in 3D4/31, ST and PK15 cells.</p

PCV2 but not PCV2 genome or PCV2-encoded components affects CSFV replication in PK15-CSFV cells.

<p>Cells were inoculated with PCV2 at MOI of 1 or 10 and pretreated with inactivated PCV2, genomic DNA of PCV2 or PCV2-encoded components. (A and C) Percentage of TUNEL-positive cells. (B and D) Titration of CSFV progeny at 1 (B) and 10 (B) MOI of BPL-inactivated PCV2. Data are represented as means ± SD (n = 3 or more; ns, <i>P</i> > 0.05; *<i>P</i> < 0.05; **<i>P</i> < 0.01).</p

Biological characteristics of PK15-CSFV cells.

<p>PK15-CSFV cells were seeded into 6- or 96-well plates and cultured for indicated time points to detect the virus titer, cell viability and apoptosis. (A) Growth curve of CSFV in PK15-CSFV cells. (B) Viability of PK15-CSFV cells by CCK–8. (C) Proportion of TUNEL-positive cells at 72 h. Data are represented as means ± SD (n = 3 or 5; ns, <i>P</i> > 0.05; *<i>P</i> < 0.05; **<i>P</i> < 0.01).</p

BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein

<p>Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection.</p