Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging

Non-enveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and human. In the picornavirus family of non-enveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses. Importance: In order to transmit their genetic material to a new host, non-enveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many non-enveloped RNA viruses the requirements for this critical part of the viral life cycle remain poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple and transferable to the study of packaging signals in other RNA viruses. Improved understanding of RNA packaging may lead to novel vaccine approaches or targets for antiviral drugs with broad spectrum activity

Mutagenesis mapping of RNA structures within the foot-and-mouth disease virus genome reveals functional elements localized in the polymerase (3Dpol)-encoding region

The Pirbright Institute receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom (projects BBS/E/I/00007035, BBS/E/I/00007036, and BBS/E/I/00007037) providing funds to cover the open access charges for this paper. This work was supported by funding from the United Kingdom Department for Environment, Food and Rural Affairs (Defra research projects SE2943 and SE2944) and BBSRC research grant BB/K003801/1.RNA structures can form functional elements that play crucial roles in the replication of positive-sense RNA viruses. While RNA structures in the untranslated regions (UTRs) of several picornaviruses have been functionally characterized, the roles of putative RNA structures predicted for protein coding sequences (or open reading frames [ORFs]) remain largely undefined. Here, we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome to predict 53 conserved RNA structures within the ORF. Forty-six of these structures were located in the regions encoding the nonstructural proteins (nsps). To investigate whether structures located in the regions encoding the nsps are required for FMDV replication, we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimize RNA secondary structures while preserving protein coding, native dinucleotide frequencies, and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV subgenomic replicon. We found that three of the RNA structures, all at the 3' termini of the FMDV ORF, were critical for replicon replication. In contrast, disruption of the other 43 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains. IMPORTANCE Some RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 46 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the nonstructural proteins, only three are essential for replication of an FMDV subgenomic replicon. Replicon replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify whether the remaining 43 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease, posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.Publisher PDFPeer reviewe

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

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

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

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 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

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 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