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. 2019 Nov 5;11(11):1030.
doi: 10.3390/v11111030.

The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

Nicole Doyle  1 , Philippa C Hawes  1 , Jennifer Simpson  1 , Lorin H Adams  1 , Helena J Maier  1
Affiliations

The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

Nicole Doyle et al. Viruses. .

Abstract

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus-host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

Keywords: DMV; coronavirus; double-membrane spherule; double-membrane vesicle; porcine deltacoronavirus; replication organelle; spherule; zippered ER.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Dynamics of porcine deltacoronavirus (PDCoV) OH-FD22 replication in LLC-PK1 cells. (A) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints, RNA was harvested and reverse transcribed to cDNA. The copy number of cDNA was quantified by quantitative polymerase chain reaction (qPCR) using a standard curve and normalized to mock-infected cells. Mean and standard deviation of three independent replicates are shown. (B) LLC-PK1 cells were PDCoV-infected or mock-infected. Total cell lysate was harvested at the stated timepoints and viral nucleoprotein detected (Anti-N) by western blot. Actin (Anti-actin) was used as a loading control. Molecular weight markers are shown. Blot representative of three independent repeats. (C) LLC-PK1 cells were infected with PDCoV and at the indicated timepoints and cell culture media was harvested. The titer of progeny virus was determined by tissue culture infectious dose 50 (TCID50). Mean and standard deviation from three independent replicates are shown.
Figure 2
Figure 2
PDCoV-associated dsRNA can be detected from 4 h post-infection (hpi). LLC-PK1 cells were infected or mock-infected. At the stated timepoints, cells were fixed and labelled with anti-dsRNA (green) and anti-N (red). Nuclei were stained with DAPI (blue) and scale bar indicates 10 μm. Images are representative of three independent replicates.
Figure 3
Figure 3
PDCoV RNA synthesis was detected from 3 hpi. LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to the indicated fixation time, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 µM actinomycin D (ActD). Mock cells were incubated with (+ActD) and without (−ActD) actinomycin D as indicated. Cells were fixed and RNA containing BrU were detected using an anti-BrdU antibody (green). Nuclei were stained with DAPI (blue), scale bar indicates 10 μm. Images representative of three independent replicates.
Figure 4
Figure 4
Sites of PDCoV RNA synthesis were associated with viral N protein, but not the endoplasmic reticulum (ER). LLC-PK1 cells were infected or mock-infected. Thirty minutes prior to fixation, cells were incubated with 2 mM 5-Bromouridine (BrU) and 15 μM actinomycin D. Cells were fixed and labelled with anti-BrdU (green) and either anti-N or anti-ER antibodies (red). Nuclei were stained with DAPI (blue), scale bar indicates 10 µm.
Figure 5
Figure 5
PDCoV RO is made up of double-membrane vesicles (DMVs) and zippered ER with double-membrane spherules. LLC-PK1 cells were mock-infected (A) or infected with PDCoV (B–F). At 8 hpi, cells were fixed with glutaraldehyde and processed for transmission electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 1 µm (A,B) or 500 nm (C–F).
Figure 6
Figure 6
DMVs and zippered ER with double-membrane spherules were present from 6 hpi to 24 hpi. LLC-PK1 cells were infected with PDCoV, and at 6 hpi (A,B) or 24 hpi (C,D), cells were fixed with glutaraldehyde and processed for electron microscopy. Virions in vesicles are indicated with black arrows, DMVs are indicated with white arrows, and regions of zippered ER with spherules are indicated with black brackets. Scale bars indicate 200 nm (A) or 500 nm (B–D).
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