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. 2014 Aug:462-463:49-59.
doi: 10.1016/j.virol.2014.04.039. Epub 2014 Jun 17.

Avian reovirus-triggered apoptosis enhances both virus spread and the processing of the viral nonstructural muNS protein

Javier Rodríguez-Grille  1 , Lisa K Busch  1 , José Martínez-Costas  1 , Javier Benavente  2
Affiliations

Avian reovirus-triggered apoptosis enhances both virus spread and the processing of the viral nonstructural muNS protein

Javier Rodríguez-Grille et al. Virology. 2014 Aug.

Abstract

Avian reovirus non-structural protein muNS is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kDa carboxyterminal protein, termed muNSC, and a 17-kDa aminoterminal polypeptide, designated muNSN. In this study we demonstrate that muNS processing is catalyzed by a caspase 3-like protease activated during the course of avian reovirus infection. The cleavage site was mapped by site directed mutagenesis between residues Asp-154 and Ala-155 of the muNS sequence. Although muNS and muNSC, but not muNSN, are able to form inclusions when expressed individually in transfected cells, only muNS is able to recruit specific ARV proteins to these structures. Furthermore, muNSC associates with ARV factories more weakly than muNS, sigmaNS and lambdaA. Finally, the inhibition of caspase activity in ARV-infected cells does not diminish ARV gene expression and replication, but drastically reduces muNS processing and the release and dissemination of progeny viral particles.

Keywords: Apoptosis; Avian reovirus; Caspase; Proteolytic processing; Viroplasms; Virus spread; muNS.

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Figures

Fig. 1
Fig. 1
Proteolytic processing of muNS occurs in ARV-infected cells, but not in transfected or baculovirus-infected cells. (A) CEF monolayers are either mock-infected (lane 1) or infected with 10 PFU/cell of ARV S1133 (lane 2) for 16 h. The same cells are transfected with either an empty pCINeo3.1 plasmid (lane 3) or with pCINeo-muNS plasmid (lane 4) for 24 h. Sf9 insect cells are infected with either wild-type baculovirus (lane 5) or with recombinant baculovirus AcNPV-S1133-muNS (lane 6) for two days. The cells in the upper panel are incubated for 1 h with 100 μCi/ml of [35S]amino acids, then lysed with RIPA buffer and immunoprecipitated with muNS-specific antiserum. An immunoblot analysis of nonradiolabeled samples is shown in the lower panel. (B) CEF monolayers are transfected with GFP-muNS for 10 h and then mock-infected (lanes 1 and 3) or infected with 10 PFU/cell of ARV S1133 for 16 h (lanes 2 and 4). The cells are then lysed in RIPA buffer and analyzed by Western blot with polyclonal antibodies against both GFP (lanes 1 and 2) and muNS (lanes 3 and 4). (C) 35S-radiolabeled in-vitro-synthesized muNS (lane 2) is incubated for 2 h at 37 °C with extracts from mock-infected cells (lane 3) or from ARV-infected cells (lane 4). These samples, as well as an immunoprecipitated extract of ARV-infected CEF (lane 1), are analyzed by 10% SDS-PAGE and autoradiography. Positions of protein markers are indicated on the left, and those of nonstructural ARV proteins on the right. The position of the muNSC band is marked with an asterisk. (D) Western blot analysis of extracts from CEF monolayers either mock-infected (lane 1) or infected for 16 h with 10 PFU/cell of the ARV isolates 1733 (lane 2), 2408 (lane 3), and S1133 (lane 4). In lane 5 the analysis is performed with an extract of Vero cells infected for 24 h with 50 PFU of ARV S1133, and in lane 6 with an extract of DF-1 cells infected for 16 h with 10 PFU of ARV S1133. The membranes in lanes 1–4 and 6 are exposed for 5 s and the one in lane 5 for 30 s.
Fig. 2
Fig. 2
Effect of apoptosis inhibitors on muNS processing. (A) Effect of apoptotic inhibitors on DNA damage. CEF monolayers are infected with 10 PFU/cell of ARV in the absence (lane 1) or presence of either 100 μM Z-VAD-FMK (Z; lane 2) or 10 μM Q-VD-OPh (Q; lane 3). Mock-infected cells either untreated (lane 4) or incubated for 6 h in the presence of 0.5 μM staurosporine (lane 5) are used as control samples. The cells are analyzed by indirect immunofluorescence with rabbit polyclonal antibodies against muNS (upper row; green), with a monoclonal antibody against phosphorylated H2AX (middle row; red), and nuclei are stained with DAPI (lower row; blue). The percentage of red-stained nuclei shown at the bottom of the figure is the mean of three independent experiments, and 100 cells were counted for each experiment. (B) Effect of the same concentrations of the two apoptotic inhibitors on caspase 3/7 activity. Caspase activity is determined with the Caspase-Glo® 3/7 Assay kit (Promega), as described in Methods section, and expressed as arbitrary RLU units. Each value is the mean of three independent experiments. (C) Effect of the two apoptotic inhibitors on muNS processing in ARV-infected CEF. Processing is monitored by both immunoprecipitation (IP; upper panel) and Western blotting (WB; lower panel) using muNS-specific antiserum. The sample in lane 1 is run on the same gel, but an internal lane is removed. (D) Effect of Q-VD-OPh on muNS processing and caspase 3/7 activity. ARV-infected CEF monolayers are incubated from the onset of the infection with the concentrations of the pancaspase inhibitor shown on top. At 16 hpi the cells are lysed and the resulting extracts are assayed for caspase 3/7 activity and muNS processing. Processing of muNS is monitored by Western blotting with polyclonal antibodies against muNS and actin. The values of caspase activity shown at the bottom of the figure are the mean of three independent experiments. (E) Time course of muNS processing and caspase 3/7 activity in ARV-infected CEF cells. CEF cell monolayers are infected with 10 PFU/cell of ARV, either in the absence (top blot) or presence (bottom blot) of 10 μM Q-VD-OPh. The cells are lysed at the infection times indicated at the top of the figure. The extracts are subsequently processed for both immunoblotting and caspase 3/7 activity, as for Fig. 2D.
Fig. 3
Fig. 3
Effect of apoptosis inhibitors/enhancers on muNS processing. (A) Effect of Q-VD-OPh on muNS processing. 35S-radiolabeled in-vitro-synthesized muNS is incubated for 4 h at 37 °C with extracts from ARV-infected cells that have been incubated (lane 2) or not (lane 1) with 10 μM Q-VD-OPh from the onset of the infection. These samples, as well as an extract of ARV-infected CEF immunoprecipitated against muNS (lane 3), are analyzed by 10% SDS-PAGE and autoradiography. (B) Effect of apoptotic enhancers and inhibitors on muNS processing and apoptosis in DF1 cells. DF-1 cell monolayers are infected with 10 PFU/cell of ARV S1133. The cells in lanes 3 and 5 are treated at the onset of the infection with 10 μM Q-VD-OPh. The cells in lanes 2 and 3 are treated from 10 to 16 hpi with 1 μg/ml actinomycin D, and in lanes 4 and 5 with 0.5 μM staurosporine. Caspase 3/7 activity, DNA damage and muNS processing are determined at 16 hpi as for Fig. 2A–C. (C) Effect of apoptotic enhancers and inhibitors on muNS processing in transfected cells. CEF monolayers are transfected with the pCINeo-muNS plasmid and 24 h later the cells are incubated for 6 h with the same compounds as for Fig. 3B. The cells are then lysed and the resulting extracts subjected to Western blot analysis with anti-muNS serum. The positions of nonstructural viral proteins are indicated on the left and that of protein markers on the right.
Fig. 4
Fig. 4
Mapping the muNS cleavage site. (A) CEF monolayers are infected with ARV S1133 (lane 1) or lipofected with plasmids expressing muNS versions comprising the residues shown on top of lanes 2–5. The cells are incubated with [35S]amino acids for 1 h at 16 hpi or for 2 h at 24 h after transfection, and then lysed with RIPA buffer. The resulting extracts are immunoprecipitated with muNS-specific antiserum and the samples are analyzed by 8% SDS-PAGE and autoradiography. The position of a non-specific protein band in transfected cells is marked with an arrow. (B) Deduced amino acid sequence of the ARV S1133 muNS region comprising residues 140–177. A potential consensus cleavage sequence for caspase 3/7 is underlined and the putative cleavage site is marked with an arrow. (C) CEF monolayers are infected with ARV S1133 (lanes 1 and 6) or transfected with empty pCINeo plasmid (lane 2) and with plasmids expressing muNS versions comprising the amino acid residues shown on top of lanes 3–5. The cells are radiolabeled with [35S]amino acids for 1 h at 16 hpi or for 5 h at 24 h after transfection, lysed with RIPA buffer and the extracts are immunoprecipitated with muNS-specific antiserum. Immunoprecipitated proteins are resolved on an 8% tricine–SDS-PAGE gel and protein bands are visualized by autoradiography. (D) The D154A muNS mutant (lane 1) and the ARV proteins shown on top (lanes 2 and 3) are transiently expressed in CEF cells, either individually (top row) or in combination with D154A (bottom row). At 24 h after transfection the cells are subjected to immunofluorescence analysis using, as primary antibodies, polyclonal antiserum against muNS (lane 1), ARV cores (lane 2), and sigmaNS (lane 3). (E) CEF monolayers are transfected with plasmids pCINeo-muNS (lanes 1–3) and pCINeo-D154A (lanes 4–5), and at 18 h post-transfection the cells in lanes 2, 3 and 5 are incubated for 6 h in the presence of 0.5 μM staurosporine, and the cells in lane 3 are also supplemented with 10 μM Q-VD-OPh. The cells are then lysed and subjected to Western blot analysis with anti-muNS serum. (F) 35S-radiolabeled in-vitro-synthesized muNS (lane 1) or its D154A point mutant (lane 2) is incubated for 4 h at 37 °C with extracts from ARV-infected cells. These samples, as well as an extract of ARV-infected CEF immunoprecipitated against muNS (lane 3), are analyzed by 10% SDS-PAGE and autoradiography. The positions of protein markers are indicated on the left and those of ARV nonstructural proteins on the right.
Fig. 5
Fig. 5
Identification of the caspase that catalyzes muNS cleavage. (A) Semiconfluent monolayers of HeLa (lanes 1–3) and MCF-7 (lanes 4–6) cells are mock-infected (lanes 1 and 4) or infected with 50 PFU/cell of ARV 1733 (lanes 2, 3, 5 and 6). The cells in lane 3 are incubated with 10 μM Q-VD-OPh from the onset of the infection and the cells in lane 6 are incubated with 0.5 μM staurosporine during the last 6 h of infection. At 16 hpi the cells are lysed in RIPA buffer and the resulting cell extracts are analyzed by Western blotting with muNS-specific antiserum. (B) 1 μg of recombinant muNS purified from baculovirus-infected insect cells (lane 2) is incubated with 1 unit of either caspase 3 (lanes 3 and 4) or caspase 7 (lanes 5 and 6) for 4 h at 37 °C, in the presence (lanes 4 and 6) or absence (lanes 3 and 5) of 10 μM Q-VD-OPh. These samples, as well as an extract of ARV-infected CEF (lane 1), are subjected to Western blot analysis using polyclonal anti-muNS serum. The positions of protein markers are shown on the left and those of nonstructural ARV proteins on the right.
Fig. 6
Fig. 6
Properties of muNS, muNSC and muNSN. (A) Plasmids expressing muNS versions comprising the amino acid residues shown on top are lipofected into CEF monolayers, and at 20 h post-transfection the cells are subjected to immunofluorescence analysis with muNS-specific antiserum as primary antibody (green), and counterstained with DAPI (blue). (B) Plasmids expressing the ARV proteins depicted on the left are transfected into CEF monolayers either individually (lane 1) or together with plasmids expressing full-length muNS (lane 2) or muNS(155–635). At 20 h post-transfection the cells are processed for immunofluorescence using as primary antibodies polyclonal antisera against ARV cores (top row) and sigmaNS (bottom row), and counterstained with DAPI (blue). (C) CEF monolayers are mock-infected (lanes 1 and 3) or infected with 10 PFU/cell of ARV S1133 (lanes 2 and 4) for 16 h. The cells are then lysed with TX-buffer, incubated for 10 min on ice and the TX-soluble fraction is removed (lanes 1 and 2). The plate-attached fraction is then solubilized with RIPA buffer (TX-insoluble fraction; lanes 3 and 4). The two fractions are analyzed by Western blotting with muNS-specific antiserum. (D) ARV-infected cells are labeled for 10 min with [35S]amino acids and then incubated in non-radioactive medium for the indicated chasing times. The cells are subsequently processed as for Fig. 2C and the resulting TX-soluble and -insoluble fractions are immunoprecipitated with muNS-specific antiserum. Immunoprecipitated proteins are resolved by electrophoresis on an 8% tricine-SDS-PAGE gel and visualized by autoradiography. Positions of protein markers are indicated on the left and those of ARV proteins on the right.
Fig. 7
Fig. 7
Effect of Q-VD-OPh on ARV replication and dissemination. (A) CEF monolayers are mock-infected (M) or infected with 10 PFU/cell of ARV S1133 (I), in the presence (+Q) or absence of 10 μM Q-VD-OPh. The cells are processed for immunoblotting at 16 hpi and the membranes are probed with rabbit polyclonal antibodies against both muNS and sigmaNS. The positions of protein markers are indicated on the left and those of the non-structural ARV proteins on the right. (B) CEF monolayers are infected with 0.1 PFU/cell of ARV S1133, and the production of both total virus (intracellular+culture medium; filled bars) and released virus (cultured medium; open bars) is determined after 24 h of infection by plaque assay on CEF monolayers. (C) Plaque pictures obtained by titrating a stock of ARV S1133 on CEF monolayers in the absence (lane 1) or presence (lane 2) of 10 μM Q-VD-OPh. The pictures in lane 1 correspond to a 106 dilution and the ones in lane 2 to 105 dilution of the same virus stock. Plaques of two different plaque assays are shown.
Fig. 8
Fig. 8
Alignment of the deduced amino acid sequences of the muNS 140–177 region from different poultry reoviruses. Sources of the host birds, virus strains, deduced amino acid sequences of the muNS region 140–177 and protein accession numbers are indicated. Putative consensus caspase cleavage sequences are boxed.
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