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

Wei T, Li D, Marcial D, Khan M, Lin MH, Snape N, Ghildyal R, Harrich D, Spann K - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Queensland Institute of Medical Research Berghofer, Herston, Australia.

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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Related in: MedlinePlus

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.
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pone-0114447-g003: 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.

Mentions: A proximity ligation assay (PLA) was performed to demonstrate the association of eEF1A with the N and P proteins of the RSV replication complex. RSV-infected A549 cells were fixed at 24 h p.i. and the PLA assay was performed using anti-eEF1A and anti-N, or anti-eEF1A and anti-P antibodies. As a control, anti-eEF1A antibody was replaced with anti-eIF3 antibody. A significant number of ligation foci for RSV N or P with eEF1A (P<0.001) were identified in infected cells, compared to ligation foci for RSV N or P with eIF3 (Figure 3A and B). The numbers of ligation foci for RSV N or P with eIF3 were similar to the un-infected cells probed with anti-eEF1A and N or P, suggesting a level of background antibody interaction (Figure 3A and B). This result suggested a strong interaction between eEF1A and the N and P proteins, most likely within RSV RNP complex in the infected cells.


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

Wei T, Li D, Marcial D, Khan M, Lin MH, Snape N, Ghildyal R, Harrich D, Spann K - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257679&req=5

pone-0114447-g003: 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.
Mentions: A proximity ligation assay (PLA) was performed to demonstrate the association of eEF1A with the N and P proteins of the RSV replication complex. RSV-infected A549 cells were fixed at 24 h p.i. and the PLA assay was performed using anti-eEF1A and anti-N, or anti-eEF1A and anti-P antibodies. As a control, anti-eEF1A antibody was replaced with anti-eIF3 antibody. A significant number of ligation foci for RSV N or P with eEF1A (P<0.001) were identified in infected cells, compared to ligation foci for RSV N or P with eIF3 (Figure 3A and B). The numbers of ligation foci for RSV N or P with eIF3 were similar to the un-infected cells probed with anti-eEF1A and N or P, suggesting a level of background antibody interaction (Figure 3A and B). This result suggested a strong interaction between eEF1A and the N and P proteins, most likely within RSV RNP complex in the infected cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Queensland Institute of Medical Research Berghofer, Herston, Australia.

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.

Show MeSH
Related in: MedlinePlus