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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

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.
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pone-0114447-g004: 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.

Mentions: In order to investigate if eEF1A is directly associated with the N and P proteins of the replication complex, co-IP assays were performed using cell lysates of both HEK293T and A549 cells infected with RSV at a MOI of 1 pfu/cell. Although reliable down-regulation of eEF1A was not achieved in A549 cells, we investigated the interactions of eEF1A and RNP complex proteins in this cell line, as a more biologically relevant cell line for RSV studies. Bound protein complexes were eluted and separated by SDS-PAGE. Western blot analysis showed that RSV N and P were co-immunoprecipitated with eEF1A in both HEK293T cells (Figure 4A) and A549 cells (Figure 4B), indicating that eEF1A effectively interacts with the RSV RNP complex in both cell lines. Interestingly, RSV M protein also bound to eEF1A in both cell types. It is likely that eEF1A interacts with N, P and M in complex during viral replication/transcription.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114447-g004: 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.
Mentions: In order to investigate if eEF1A is directly associated with the N and P proteins of the replication complex, co-IP assays were performed using cell lysates of both HEK293T and A549 cells infected with RSV at a MOI of 1 pfu/cell. Although reliable down-regulation of eEF1A was not achieved in A549 cells, we investigated the interactions of eEF1A and RNP complex proteins in this cell line, as a more biologically relevant cell line for RSV studies. Bound protein complexes were eluted and separated by SDS-PAGE. Western blot analysis showed that RSV N and P were co-immunoprecipitated with eEF1A in both HEK293T cells (Figure 4A) and A549 cells (Figure 4B), indicating that eEF1A effectively interacts with the RSV RNP complex in both cell lines. Interestingly, RSV M protein also bound to eEF1A in both cell types. It is likely that eEF1A interacts with N, P and M in complex during viral replication/transcription.

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