PDI and ERp57 appear at the surface of NSDV-infected cells.

<p><b>A.</b> Samples were prepared as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094656#pone-0094656-g001" target="_blank">Figure 1</a> except that, after fixing with 3% PFA, cells were labelled with mouse anti-PDI (clone 1D3; a–c), mouse anti-PDI (clone RL90; d–f) or rabbit anti-ERp57 (g–i). Then cells were again fixed with 3% PFA, opened with ice-cold methanol, and stained with rabbit antiserum against the viral N protein (a–f) or with mouse anti-PreGn antibody (g–i). Proteins were visualised by co-staining with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). <b>B.</b> Vero cells were infected with the NSDVi isolate at a MOI of 6 TCID₅₀ or left uninfected. After 16 h, cells were fixed using 3% PFA and left non-permeabilised. Cells were incubated with mouse anti-PDI (clone 1D3) antibody followed by Alexa Fluor 488 goat anti-mouse IgG (green). Nuclei were counterstained using DAPI (blue). Bars correspond to 40 μm.</p

NSDV replication has no obvious effect on the ERGIC and Golgi.

<p>Vero cells were infected with the NSDVi isolate at a MOI of 0.3 TCID₅₀. After 16 h, cells were fixed using 3% PFA followed by ice-cold methanol. Cells were co-stained with mouse anti-ERGIC53 (ERGIC; a–c), mouse anti-GM130 (<i>cis</i>Golgi; d–f) or rat anti-p102 (<i>trans</i>Golgi; g–i) and rabbit antiserum against the viral N protein. Proteins were visualised by co-staining with Alexa Fluor 488 goat anti-mouse or anti-rat IgG (green), and Alexa Fluor 568 goat anti-rabbit IgG (red). Nuclei were counterstained using DAPI (blue). Bars correspond to 20 μm.</p

The replication of NSDV induces secretion of PDI and ERp57 from infected cells.

<p>Vero cells were infected with the NSDVi isolate at a MOI of 0.1 TCID₅₀ or left uninfected. After 48 h, supernatants and cells were harvested; proteins were separated on acrylamide SDS-PAGE gels and cellular and viral proteins were detected by Western blotting using specific antibodies to PDI (clone RL90), ERp57, the viral N protein, calnexin (CNX) or α-tubulin.</p

Viral glycoprotein association with PDI and ERp57.

<p><b>A.</b> Samples were prepared as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094656#pone-0094656-g001" target="_blank">Figure 1</a> except that cells were co-stained with rabbit anti-ERp57 and mouse anti-PreGn antibodies followed by co-staining with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). Bar corresponds to 40 μm. <b>B.</b> Vero cells were infected with the NSDVi isolate at a MOI of 0.1 TCID₅₀ or left uninfected. After 48 h, cells were harvested and proteins were immunoprecipitated (IP) from cell lysates using mouse anti-PDI (clone RL90) antibody and protein G-agarose beads. Proteins were separated on acrylamide SDS-PAGE gels and detected by Western blotting (WB) using anti-PreGn, anti-PDI or anti-N antibodies.</p

The Nairovirus Nairobi Sheep Disease Virus/Ganjam Virus Induces the Translocation of Protein Disulphide Isomerase-Like Oxidoreductases from the Endoplasmic Reticulum to the Cell Surface and the Extracellular Space

<div><p>Nairobi sheep disease virus (NSDV) of the genus <i>Nairovirus</i> causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI), an oxidoreductase present in the lumen of the endoplasmic reticulum (ER) and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.</p></div

PDI and ERp57 appear not to be degraded in NSDV-infected cells.

<p>Vero cells were infected with the NSDVi isolate at a MOI of 5 TCID₅₀ or left uninfected. After 16 h, cells were harvested by lysis and proteins separated on an acrylamide SDS-PAGE gel; proteins were detected by Western blot using antibodies specific to PDI (clone RL90), ERp57, calnexin (CNX), <i>cis</i> Golgi (GM130), <i>trans</i> Golgi (p102), PCNA and the NSDV N protein.</p

NSDV-infected cells show reduced level of PDI and ERp57 in the ER.

<p>The experiment was carried out as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094656#pone-0094656-g001" target="_blank">Figure 1</a>, but cells were co-stained with mouse anti-PDI (clone 1D3; a–c), mouse anti-PDI (clone RL90; d–f), rabbit anti-ERp57 (g–i) or mouse anti-calnexin (j–l), and rabbit antiserum against the viral N protein. Proteins were subsequently visualised by co-staining with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red) (a–f and j–l), except for g–i, where rabbit anti-ERp57 IgGs were labelled with Zenon Alexa Fluor 488 rabbit IgG labelling reagent (green) and rabbit anti-N IgGs were labelled with Zenon Alexa Fluor 594 rabbit IgG labelling reagent (red). Bars correspond to 40 μm.</p

The effect of NSDV glycoprotein expression on PDI.

<p>Vero cells were transfected with 1 μg of pCAGGs_MCSII_PreGn_V5 (a–c and j–l), pCAGGs_MCSII_PreGc_V5 (d–f) or pCAGGs_MCSII_NS_M_V5 (g–i). After 24 h, cells were fixed with 3% PFA followed by ice-cold methanol, and were stained using mouse anti-PDI (a–i) or anti-calnexin (j–l) antibodies followed by Alexa Fluor 568 goat anti-mouse IgG (red). Plasmid-expressed proteins were visualised with mouse anti-V5 antibody conjugated to Alexa Fluor 488 (green). Nuclei were counterstained using DAPI (blue). Bars correspond to 40 μm. Arrows in a–b and j–k indicate cells expressing PreGn.</p

Time course of changes to PDI in NSDV-infected cells.

<p>Vero cells were infected with the NSDVi isolate at a MOI of 6 TCID₅₀ and fixed at 8, 12 and 16 hpi. For internal staining (a–i) cells were fixed using 3% PFA followed by ice cold methanol. Cells were co-stained with mouse anti-PDI (clone 1D3) antibody and rabbit antiserum against the viral N protein. For surface PDI staining (j–l), cells were fixed with 3% PFA followed by staining with mouse anti-PDI (clone 1D3) antibody. Then cells were again fixed with 3% PFA, opened with ice-cold methanol, and virus was detected by rabbit antiserum against the N protein. Proteins were visualised with Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). Nuclei were counterstained using DAPI (blue). Bars correspond to 16 μm.</p

Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

<div><p>Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus <i>Nairovirus</i>. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus <i>in vivo</i>. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.</p></div