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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

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
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pone-0114447-g001: 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

Mentions: We previously used siRNA to down-regulate eEF1A in HIV-1 infected cells to demonstrate that eEF1A is a component of the HIV-1 reverse transcriptase complex and an important factor in HIV-1 early replication [19]. We used the same siRNA (called sieEF1A), or a mismatch siRNA (siMM) molecule as a negative control, to down-regulate eEF1A in HEK293T and A549 cells. Down-regulation of eEF1A was limited in A549 cells, hence HEK293T cells were used in this study. Transfected and untransfected HEK293T cells were infected with RSV at a MOI of 0.1 pfu/cell 48 h following transfection. The amount of infectious virus shed into cell culture supernatant over the subsequent 48 h was quantified by performing immune-plaque assay for three independent experiments. Down-regulation of eEF1A resulted in a significant (P<0.05) reduction of viral titres 48 h post-infection (p.i.) compared to cells either not transfected or transfected with siMM (Figure 1A). While siMM or untransfected cells shed 10-fold more RSV at 48 h compared to 2 h post-infection, sieEF1A-transfected cells shed very little virus. Western blot analysis demonstrated that eEF1A down-regulation was sustained for the 48 h p.i. (96 h post-transfection; Figure 1B and 1C). Cell viability was also not affected by transfection with either sieEF1A or siMM, compared to non-transfected cells at 72 and 96 h post transfection (Figure 1D). Although the amount of virus shed from infected and untreated HEK293T cells is less that would be expected from A549 cells [17] these data do demonstrates a biological dependency of RSV on eEF1A for the efficient shedding of infectious virus.


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)

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
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pone-0114447-g001: 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
Mentions: We previously used siRNA to down-regulate eEF1A in HIV-1 infected cells to demonstrate that eEF1A is a component of the HIV-1 reverse transcriptase complex and an important factor in HIV-1 early replication [19]. We used the same siRNA (called sieEF1A), or a mismatch siRNA (siMM) molecule as a negative control, to down-regulate eEF1A in HEK293T and A549 cells. Down-regulation of eEF1A was limited in A549 cells, hence HEK293T cells were used in this study. Transfected and untransfected HEK293T cells were infected with RSV at a MOI of 0.1 pfu/cell 48 h following transfection. The amount of infectious virus shed into cell culture supernatant over the subsequent 48 h was quantified by performing immune-plaque assay for three independent experiments. Down-regulation of eEF1A resulted in a significant (P<0.05) reduction of viral titres 48 h post-infection (p.i.) compared to cells either not transfected or transfected with siMM (Figure 1A). While siMM or untransfected cells shed 10-fold more RSV at 48 h compared to 2 h post-infection, sieEF1A-transfected cells shed very little virus. Western blot analysis demonstrated that eEF1A down-regulation was sustained for the 48 h p.i. (96 h post-transfection; Figure 1B and 1C). Cell viability was also not affected by transfection with either sieEF1A or siMM, compared to non-transfected cells at 72 and 96 h post transfection (Figure 1D). Although the amount of virus shed from infected and untreated HEK293T cells is less that would be expected from A549 cells [17] these data do demonstrates a biological dependency of RSV on eEF1A for the efficient shedding of infectious virus.

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