Limits...
mRNA decay factor AUF1 binds the internal ribosomal entry site of enterovirus 71 and inhibits virus replication.

Lin JY, Li ML, Brewer G - PLoS ONE (2014)

Bottom Line: During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells.AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins.Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.

ABSTRACT
AU-rich element binding factor 1 (AUF1) has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES) of enterovirus 71 (EV71) and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

Show MeSH

Related in: MedlinePlus

AUF1 interacts with the EV71 5′UTR.(A) RNA-protein pull-down experiments were performed to examine the interaction between AUF1 and the EV71 5′UTR. The EV71 5′UTR was synthesized in vitro in the presence of biotin-UTP. Biotinylated RNA was incubated with SF268 cell lysate (200 µg proteins) for 15 min at 30°C. Unlabeled RNA was added to parallel reactions as a negative control. Streptavidin-linked beads were used to pull-down biotin-labeled EV71 5′UTR and its associated cellular proteins. The beads were washed and then resuspended in SDS-PAGE loading buffer to dissociate proteins from RNA. Samples were boiled and analyzed by Western blot using anti-AUF1 antibody. (B) Secondary structures within the 5′UTR were predicted by MFold. Numbers below each stem-loop indicate 5′UTR nucleotides encompassing the respective stem-loop. RNA substrates used for protein-RNA pull-down experiments contained the indicated stem-loop and the flanking region immediately 5′ to it. (C) Identification of AUF1 interaction site(s) within the EV71 5′UTR. Biotinylated RNAs containing the indicated stem-loops were synthesized; control RNAs lacked biotin. SL-VI extends to nt 745, so it contains linker sequence between the IRES and the AUG. RNA-protein pull-downs and Western blot analysis were carried out as described for panel (A). The blot was stripped and re-probed with anti-FBP1 antibody as a positive control for RNA-binding activity.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4117571&req=5

pone-0103827-g002: AUF1 interacts with the EV71 5′UTR.(A) RNA-protein pull-down experiments were performed to examine the interaction between AUF1 and the EV71 5′UTR. The EV71 5′UTR was synthesized in vitro in the presence of biotin-UTP. Biotinylated RNA was incubated with SF268 cell lysate (200 µg proteins) for 15 min at 30°C. Unlabeled RNA was added to parallel reactions as a negative control. Streptavidin-linked beads were used to pull-down biotin-labeled EV71 5′UTR and its associated cellular proteins. The beads were washed and then resuspended in SDS-PAGE loading buffer to dissociate proteins from RNA. Samples were boiled and analyzed by Western blot using anti-AUF1 antibody. (B) Secondary structures within the 5′UTR were predicted by MFold. Numbers below each stem-loop indicate 5′UTR nucleotides encompassing the respective stem-loop. RNA substrates used for protein-RNA pull-down experiments contained the indicated stem-loop and the flanking region immediately 5′ to it. (C) Identification of AUF1 interaction site(s) within the EV71 5′UTR. Biotinylated RNAs containing the indicated stem-loops were synthesized; control RNAs lacked biotin. SL-VI extends to nt 745, so it contains linker sequence between the IRES and the AUG. RNA-protein pull-downs and Western blot analysis were carried out as described for panel (A). The blot was stripped and re-probed with anti-FBP1 antibody as a positive control for RNA-binding activity.

Mentions: Binding of AUF1 to enterovirus RNA was first described by Lenarcic et al. [41]. As noted above, Semler and colleagues showed that AUF1 associates with the IRES of enterovirus and human rhinovirus. The EV71 5′UTR plays roles in viral translation and replication. To determine whether AUF1 interacts with the EV71 5′UTR, the full-length EV71 5′UTR was synthesized in vitro with biotin-UTP. The RNA was mixed with 200 µg of proteins from SF268 cell extract. Streptavidin beads were used to capture biotinylated EV71 5′UTR bound to cellular proteins. Western blotting was carried out to detect AUF1. Figure 2A shows that the indicated AUF1 isoforms associated with the EV71 5′UTR. No pull-down of AUF1 was observed when unlabeled 5′UTR was used.


mRNA decay factor AUF1 binds the internal ribosomal entry site of enterovirus 71 and inhibits virus replication.

Lin JY, Li ML, Brewer G - PLoS ONE (2014)

AUF1 interacts with the EV71 5′UTR.(A) RNA-protein pull-down experiments were performed to examine the interaction between AUF1 and the EV71 5′UTR. The EV71 5′UTR was synthesized in vitro in the presence of biotin-UTP. Biotinylated RNA was incubated with SF268 cell lysate (200 µg proteins) for 15 min at 30°C. Unlabeled RNA was added to parallel reactions as a negative control. Streptavidin-linked beads were used to pull-down biotin-labeled EV71 5′UTR and its associated cellular proteins. The beads were washed and then resuspended in SDS-PAGE loading buffer to dissociate proteins from RNA. Samples were boiled and analyzed by Western blot using anti-AUF1 antibody. (B) Secondary structures within the 5′UTR were predicted by MFold. Numbers below each stem-loop indicate 5′UTR nucleotides encompassing the respective stem-loop. RNA substrates used for protein-RNA pull-down experiments contained the indicated stem-loop and the flanking region immediately 5′ to it. (C) Identification of AUF1 interaction site(s) within the EV71 5′UTR. Biotinylated RNAs containing the indicated stem-loops were synthesized; control RNAs lacked biotin. SL-VI extends to nt 745, so it contains linker sequence between the IRES and the AUG. RNA-protein pull-downs and Western blot analysis were carried out as described for panel (A). The blot was stripped and re-probed with anti-FBP1 antibody as a positive control for RNA-binding activity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117571&req=5

pone-0103827-g002: AUF1 interacts with the EV71 5′UTR.(A) RNA-protein pull-down experiments were performed to examine the interaction between AUF1 and the EV71 5′UTR. The EV71 5′UTR was synthesized in vitro in the presence of biotin-UTP. Biotinylated RNA was incubated with SF268 cell lysate (200 µg proteins) for 15 min at 30°C. Unlabeled RNA was added to parallel reactions as a negative control. Streptavidin-linked beads were used to pull-down biotin-labeled EV71 5′UTR and its associated cellular proteins. The beads were washed and then resuspended in SDS-PAGE loading buffer to dissociate proteins from RNA. Samples were boiled and analyzed by Western blot using anti-AUF1 antibody. (B) Secondary structures within the 5′UTR were predicted by MFold. Numbers below each stem-loop indicate 5′UTR nucleotides encompassing the respective stem-loop. RNA substrates used for protein-RNA pull-down experiments contained the indicated stem-loop and the flanking region immediately 5′ to it. (C) Identification of AUF1 interaction site(s) within the EV71 5′UTR. Biotinylated RNAs containing the indicated stem-loops were synthesized; control RNAs lacked biotin. SL-VI extends to nt 745, so it contains linker sequence between the IRES and the AUG. RNA-protein pull-downs and Western blot analysis were carried out as described for panel (A). The blot was stripped and re-probed with anti-FBP1 antibody as a positive control for RNA-binding activity.
Mentions: Binding of AUF1 to enterovirus RNA was first described by Lenarcic et al. [41]. As noted above, Semler and colleagues showed that AUF1 associates with the IRES of enterovirus and human rhinovirus. The EV71 5′UTR plays roles in viral translation and replication. To determine whether AUF1 interacts with the EV71 5′UTR, the full-length EV71 5′UTR was synthesized in vitro with biotin-UTP. The RNA was mixed with 200 µg of proteins from SF268 cell extract. Streptavidin beads were used to capture biotinylated EV71 5′UTR bound to cellular proteins. Western blotting was carried out to detect AUF1. Figure 2A shows that the indicated AUF1 isoforms associated with the EV71 5′UTR. No pull-down of AUF1 was observed when unlabeled 5′UTR was used.

Bottom Line: During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells.AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins.Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.

ABSTRACT
AU-rich element binding factor 1 (AUF1) has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES) of enterovirus 71 (EV71) and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

Show MeSH
Related in: MedlinePlus