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. 2005 Jul;79(13):8090-100.
doi: 10.1128/JVI.79.13.8090-8100.2005.

Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability

Jayme Salsman  1 , Deniz Top, Julie Boutilier, Roy Duncan
Affiliations

Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability

Jayme Salsman et al. J Virol. 2005 Jul.

Abstract

The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by non-enveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively non-leaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ecto-domain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic non-enveloped viruses, with non-leaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.

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Figures

FIG. 1.
FIG. 1.
FAST protein structural motifs. The linear arrangement of structural motifs present in the p10 (ARV or NBV), BRV p15, and RRV p14 reovirus FAST proteins is depicted. HP, hydrophobic patch; TM, transmembrane domain; PB, polybasic region; myr, myristoylation; pal, palmitoylation; PP, polyproline; C, cysteine residue.
FIG. 2.
FIG. 2.
Increased membrane permeability during late-stage avian reovirus infection correlates with virus release. (A) QM5 cells preloaded with [3H]uridine were infected with ARV-138 (MOI = 5) and harvested at the indicated times postinfection. Percent uridine release from mock- and ARV-138-infected cells was quantified by scintillation counting, and the percent extracellular virus was determined by plaque assay. Results are expressed as the means ± standard deviations of a representative experiment conducted in triplicate. (B) Infected cells were fixed and Giemsa stained at the indicated times postinfection to detect syncytium formation. Images were captured by light microscopy at ×100 magnification. The arrow indicates an early syncytium.
FIG. 3.
FIG. 3.
Syncytium formation induced by the p10 FAST proteins results in increased late-stage membrane permeability. (A) QM5 cells were labeled with [3H]uridine prior to transfection with the control plasmid (mock) or with plasmids expressing the NBV p10 or ARV p10 FAST protein. At the indicated times posttransfection, the percent uridine release was quantified by scintillation counting. Data are presented as the means ± standard errors of three independent experiments. At the same time points, parallel Giemsa-stained monolayers were observed by light microscopy and qualitatively scored for the extent of syncytium formation on a scale of 0 to 4. (B) Light microscopy images (×80 magnification) of Giemsa-stained monolayers transfected with the NBV p10 expression plasmid at various times (hours) posttransfection (hpt). The SI at each time point is indicated and corresponds to the results presented in panel A. The same procedure was used to determine the SI of ARV p10.
FIG. 4.
FIG. 4.
Membrane permeability changes induced by the BRV p15 and RRV p14 FAST proteins. (A) QM5 cells were labeled with [3H]uridine prior to transfection with the control plasmid (mock) or with plasmids expressing the BRV p15 or RRV p14 FAST protein. At the indicated times posttransfection, the percent uridine release was quantified by scintillation counting. Data are presented as the means ± standard errors of three independent experiments. The syncytial index was determined at the same time points, as described in the legend of Fig. 3. (B) Light microscopy images (×80 magnification) of Giemsa-stained monolayers transfected with the BRV p15 expression plasmid at various times (hours) posttransfection (hpt). The SI at each time point is indicated. The same procedure was used to determine the SI of RRV p14.
FIG. 5.
FIG. 5.
Inhibition of syncytium formation prevents membrane permeabilization. (A) QM5 cells were labeled with [3H]uridine prior to transfection with the empty vector (mock) or with the RRV p14 expression vector. At 3 h posttransfection, anti-p14 polyclonal antiserum (α-p14) or normal rabbit serum (NRS) was added to cells (1:20 dilution). The percent uridine release and syncytial index were determined at 20 h posttransfection, as described in the legend of Fig. 3. Values represent the means ± standard deviations of a representative experiment conducted in triplicate. (B) QM5 cells were labeled with [3H]uridine prior to transfection with the empty vector (mock), with a plasmid vector expressing the authentic ARV p10 protein (p10), or with a plasmid expressing a nonfusogenic N-terminally-truncated version the ARV p10 protein (p10-del). The percent uridine release and syncytial index were determined at 48 h posttransfection, as described in the legend of Fig. 3. Values represent the means ± standard deviations of a representative experiment conducted in triplicate.
FIG. 6.
FIG. 6.
Membrane permeabilization is bidirectional and not cell specific. QM5 (panels A and B) or Vero cells (panel C) were transfected with empty vector (mock) or with vectors expressing the RRV p14 or NBV p10 FAST protein. At the indicated times (hours) posttransfection (hpt), cells were incubated in methionine-free medium in the absence or presence of 1.5 mM hygromycin B for 45 min and then pulse-labeled with [35S]methionine with or without hygromycin B for 45 min. Cells were lysed in radioimmunoprecipitation buffer, and radiolabeled cell proteins were resolved by SDS-polyacrylamide gel electrophoresis (15% acrylamide) and detected by autoradiography to assess the relative degree of translation in cells. The SI at the time of labeling was determined by Giemsa staining and light microscopy, as described in the legend of Fig. 3. RRV* denotes RRV p14-transfected cells treated with anti-p14 antibody to inhibit fusion, as described in the legend of Fig. 5.
FIG. 7.
FIG. 7.
FAST-mediated syncytium formation triggers apoptosis. (A) QM5 cells transfected with empty vector (mock) or an RRV p14 expression vector were fixed at 24 h posttransfection and stained with DAPI. Arrows in the right panel indicate the edges of a single syncytium containing a cluster of pyknotic nuclei. Scale bar = 10 μm. (B) DNA was isolated from mock-transfected cells or from transfected cells expressing the indicated FAST proteins at various times (hours) posttransfection (hpt). One of the p14-transfected samples was incubated with anti-p14 antiserum to inhibit syncytium formation, as described in the legend of Fig. 5. Isolated DNA was resolved on 1% agarose gels. The locations of intact (*) and oligonucleosomal DNA fragments (**) are indicated on the right. The locations of DNA size markers are indicated on the left. The SI at the time of DNA isolation was determined as described in the legend of Fig. 3 and is indicated for each sample.
FIG. 8.
FIG. 8.
Inhibiting caspase activity inhibits increased membrane permeability. (A) QM5 cells were labeled with [3H]uridine prior to transfection with the empty vector (mock) or with the RRV p14 expression vector. Cells were incubated in the absence or presence of the pancaspase inhibitor Z-Vad-fmk. The percent uridine release was determined at the indicated times posttransfection, as described in the legend of Fig. 3. Values represent the means ± standard deviations of a representative experiment conducted in triplicate. Numbers within each histogram indicate the syncytial index of each sample at that time point, determined as described in the legend of Fig. 3. (B) DNA was isolated from mock-transfected or p14-transfected cells at 16 h posttransfection. Isolated DNA was resolved on 1% agarose gels. The locations of DNA size markers are indicated on the left. The SI at the time of DNA isolation was determined as described in the legend of Fig. 3 and is indicated for each sample. (C) Cells expressing p14 in the absence or presence of Z-Vad-fmk were fixed and Giemsa stained at 16 h posttransfection, at which time the entire monolayer was fused and contained large clusters of nuclei present in a continuous syncytium. Arrows in the left panel indicate the edges of the syncytium detaching from the substratum.
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