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. 2014 Dec 5;9(12):e114447.
doi: 10.1371/journal.pone.0114447. eCollection 2014.

The eukaryotic elongation factor 1A is critical for genome replication of the paramyxovirus respiratory syncytial virus

Ting Wei  1 , Dongsheng Li  1 , Daneth Marcial  2 , Moshin Khan  3 , Min-Hsuan Lin  1 , Natale Snape  4 , Reena Ghildyal  5 , David Harrich  6 , Kirsten Spann  7
Affiliations

The eukaryotic elongation factor 1A is critical for genome replication of the paramyxovirus respiratory syncytial virus

Ting Wei et al. PLoS One. .

Abstract

The eukaryotic translation factor eEF1A assists replication of many RNA viruses by various mechanisms. Here we show that down-regulation of eEF1A restricts the expression of viral genomic RNA and the release of infectious virus, demonstrating a biological requirement for eEF1A in the respiratory syncytial virus (RSV) life cycle. The key proteins in the replicase/transcriptase complex of RSV; the nucleocapsid (N) protein, phosphoprotein (P) and matrix (M) protein, all associate with eEF1A in RSV infected cells, although N is the strongest binding partner. Using individually expressed proteins, N, but not P or M bound to eEF1A. This study demonstrates a novel interaction between eEF1A and the RSV replication complex, through binding to N protein, to facilitate genomic RNA synthesis and virus production.

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Conflict of interest statement

Competing Interests: Co-author David Harrich is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to PLOS ONE editorial policies and criteria.

Figures

Figure 1
Figure 1. Down-regulation of eEF1A reduces the amount of infectious virus released from RSV-infected cells.
HEK293T cells were transfected with sieEF1A, or a non-specific control siRNA (siMM), 48 h prior to infection with RSV A2 at a MOI of 0.1 pfu/cell. Infectious virus released from the cells into the culture supernatant was quantified by immune-plaque assay using Hep-2a cells exposed to culture supernatants, then overlayed with methyl cellulose and incubated at 37°C for 6 days. RSV-positive plaques were detected using antisera to RSV. (A) The amount of infectious virus released by cells in which eEF1A had been down-regulated was significantly reduced 48 h post-infection, compared to cells that had been transfected with the siMM control or untreated. (B) Down-regulation of eEF1A>90% by sieEF1A and not the siMM control was confirmed by western blot analysis of cell lysates at the time of infection, which was 48 h after transfection and also for the 24 h and 48 h following RSV infection (72 h and 96 h post-transfection). Beta-Tubulin was used as a loading control. (C) A digitized western blot in (B) was analyzed using ImageJ software. The eEF1A level (average pixel intensity) in each lane was normalized to the corresponding level of β-tubulin in the same lane. For each time point, the amount of eEF1A detected in untreated control samples was designated as 100%. (D) HEK293T cells transfected with either sieEF1A or siMM, remained viable compared to untreated cells 72 h and 96 h post-transfection. The average cell viability compared with an uninfected control was calculated. siRNA experiments were repeated three times with similar results. Mean values, with SEM are shown. Significance identified using paired t-test. *P<0.05
Figure 2
Figure 2. Down-regulation of eEF1A reduces RSV genome expression, but not mRNA transcription or protein expression.
HEK273T cells were infected with RSV at a MOI of 0.1 pfu/cell. (A) The transcription of RSV nucleocapsid (N) mRNA, was quantified by qRT-PCR using dual-labelled probes specific for RSV N and β-actin. N mRNA was expressed as fold induction compared to uninfected cells, and normalized to β-actin expression. Down-regulation of eEF1A did not affect RSV N mRNA transcription. (B) Down-regulation of eEF1A significantly reduced replication of RSV genomic RNA, as quantified by qRT-PCR using dual-labeled probes specific for the N-P (phosphoprotein) region of the RSV genome and β-actin. RSV genomic RNA was expressed as fold induction compared to uninfected cells, normalized to β-actin expression. (C) The ratio of viral mRNA/genomic RNA was calculated using the same data as in A and B, and identified a trend towards elevated viral mRNA relative to genomic RNA when eEF1A is down-regulated. (D) RSV protein expression was not affected by down-regulation of eEF1A, as demonstrated by Western blot analysis of SDS-PAGE separated proteins using an anti-RSV polyclonal antibody that detected RSV N, P and M. β-tubulin was used as a loading control. (E) Densitometry was performed using the Odyssey (Li-Cor) to quantify signal intensity of the RSV N, P and M proteins detected by western blot analysis and normalised against β-tubulin expression. Experiments were repeated three times. Mean values, with SEM are shown. Significance identified using paired t-test. *P<0.05
Figure 3
Figure 3. Nucleocapsid (N) and phosphoprotein (P) co-localise with host eEF1A in a live virus infection.
A549 cells were infected with RSV at a MOI of 1 pfu/cell and fixed for proximity assay 24 h post-infection. Antibodies to eEF1A or eIF3A (negative control), and RSV N and P were used in conjunction with Duolink II PLA probes to detect significant proximity between RSV N, P and eEF1A, and no significant proximity between RSV N, P and eIF3A. (A) Images were captured on a confocal microscope and (B) the number of signals (foci) detected in 100 cells per reaction was quantified using the Duolink Imagetool software (Olink Biosciences). Data was collected by two readers and the mean number of signals/cell was then calculated. Experiments were repeated three times. Mean values, with SEM. Significance identified using 2-way ANOVA. **P<0.05, ***P<0.001.
Figure 4
Figure 4. RSV nucleocapsid (N), phosphoprotein (P) and matrix (M) bind to eEF1A in a live virus infection.
(A) HEK293T or (B) A549 cells were infected with RSV at a MOI of 1 pfu/cell and lysed 48 h post-infection. The lysate was incubated with beads bound with antibodies to either eEF1A or eIF3A (negative control). Western blot analysis was performed on lysates before and after immunoprecipitation using antibodies to RSV or eEF1A. A representative blot of immunoprecipitation performed 3 times with consistent results is shown.
Figure 5
Figure 5. Nucleocapsid (N), but not phosphoprotein (P) or matrix (M) protein bind to eEF1A when expressed singly from plasmids.
BSR-T7 cells were transfected with plasmids expressing either (A) RSV N or (B) RSV P and immunoprecipitation performed using beads either unbound (beads only) or bound with antibodies to eEF1A. Lysates before and after immunoprecipitation were separated by SDS-PAGE and probed using a polyclonal anti-RSV antibody and an anti-eEF1A antibody. (C) HEK293T cells were transfected with a plasmid expressing FLAG-M and immunoprecipitation performed using beads either unbound (beads only) or bound with antibodies to FLAG. Lysates before and after immunoprecipitation were separated by SDS-PAGE and probed using an anti-RSV polyclonal antibody, anti-FLAG antibody, or anti-eEF1A antibody.
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Grants and funding

This study was funded by the Australian National Health and Medical Research Council Project Grant #1065121 to DH and KS (https://www.nhmrc.gov.au/); Australian Infectious Diseases Research Centre project grant to KS and DH (www.aidrc.org.au/); Australian National Health and Medical Research Council Biomedical Early Career Development Fellowship to TW; and Australian Research Council Future Fellowship to DH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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