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 can compete with hnRNP A1 for association with the IRES.(A) The pull-down assay was performed with SF268 lysate and biotin-labeled EV71 5′UTR as described for Figure 2. Additionally, increasing amounts of purified recombinant p40AUF1 were added to reaction mixtures to examine the effects on hnRNP A1 binding to biotin-labeled EV71 5′UTR. Eluted proteins were analyzed by Western blot using anti-hnRNP A1 antibody (upper panel) or AUF1 antibody (lower panel). Lane 1: non-biotinylated RNA was added to the reaction as a negative control. Lanes 3–6: 0.2, 0.5, 1, and 1.5 µg of purified p40AUF1 was added to reactions, respectively. (B) The pull-down assay was performed as described in panel (A) except increasing amounts of purified recombinant FBP1 were added to the reaction mixtures. (C) Biotin-labeled SL-II was used in the pull-down assay to examine the effect of increasing amounts of p40AUF1 on hnRNP A1–SL-II interactions. (D) Biotin-labeled SL-II and increasing amounts of FBP1 were used in the pull-down assay to assess the effect of FBP1 on hnRNP A1–SL-II interactions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103827-g005: AUF1 can compete with hnRNP A1 for association with the IRES.(A) The pull-down assay was performed with SF268 lysate and biotin-labeled EV71 5′UTR as described for Figure 2. Additionally, increasing amounts of purified recombinant p40AUF1 were added to reaction mixtures to examine the effects on hnRNP A1 binding to biotin-labeled EV71 5′UTR. Eluted proteins were analyzed by Western blot using anti-hnRNP A1 antibody (upper panel) or AUF1 antibody (lower panel). Lane 1: non-biotinylated RNA was added to the reaction as a negative control. Lanes 3–6: 0.2, 0.5, 1, and 1.5 µg of purified p40AUF1 was added to reactions, respectively. (B) The pull-down assay was performed as described in panel (A) except increasing amounts of purified recombinant FBP1 were added to the reaction mixtures. (C) Biotin-labeled SL-II was used in the pull-down assay to examine the effect of increasing amounts of p40AUF1 on hnRNP A1–SL-II interactions. (D) Biotin-labeled SL-II and increasing amounts of FBP1 were used in the pull-down assay to assess the effect of FBP1 on hnRNP A1–SL-II interactions.

Mentions: One mechanism that might account for how AUF1 could reduce IRES activity is that it competes with positive-acting ITAFs for binding to the EV71 5′UTR, thereby reducing their access to the IRES. Our previous work demonstrated that hnRNP A1 associates with SL-II within the EV71 5′UTR and promotes viral translation and replication [16][43]. AUF1 also associates with SL-II (Fig. 2C). AUF1 and hnRNP A1 are homologous in amino acid sequence and functional domains, particularly in the two RRMs (RNA recognition motifs) and C-terminal glycine-rich region (see Supplementary Fig. 1 in [44]). To determine whether AUF1 and hnRNP A1 might compete for binding to the EV71 5′UTR, a protein-RNA pull-down and competition assay was performed. Biotinylated 5′UTR and 200 µg of protein from cell lysates were mixed in reactions that included increasing amounts of purified recombinant His6-p40AUF1. This isoform was chosen since it is predominantly cytoplasmic [45], and it is expressed in SF268 cells. Binding was measured by protein-RNA pull-down followed by Western blotting, similar to Figure 2. As shown in Figure 5A, the amount of hnRNP A1 that was RNA-associated decreased with increasing amounts of p40AUF1 added; binding by hnRNP A1 was reduced 73% with the maximum p40AUF1 added. As expected, increasing p40AUF1 in reactions led to its increased association with RNA. As a control for specificity, effects of added FBP1 on binding by hnRNP A1 were examined (Fig. 5B). The amount of hnRNP A1 pulled-down by biotin-5′UTR remained constant despite addition of increasing amounts of FBP1. This is because FBP1 binds the linker region (nt 637–745) of the 5′UTR, while hnRNP A1 binds SL-II [13]. To determine if AUF1 and hnRNP A1 compete for 5′UTR binding via SL-II, biotin-labeled SL-II was used in the RNA pull-down assay. As shown in Figure 5C, the amount of hnRNP A1 associated with SL-II decreased with increasing amounts of p40AUF1 added; binding by hnRNP A1 was decreased by 80% with the maximum amount of p40AUF1 added. By contrast, the amount of hnRNP A1 associated with SL-II remained constant with increasing amounts of FBP1 added; SL-II did not pull-down FBP1, since it does not bind SL-II, as noted above (Fig. 5D). Taken together, these results suggest reciprocal binding of SL-II by AUF1 and hnRNP A1.


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 can compete with hnRNP A1 for association with the IRES.(A) The pull-down assay was performed with SF268 lysate and biotin-labeled EV71 5′UTR as described for Figure 2. Additionally, increasing amounts of purified recombinant p40AUF1 were added to reaction mixtures to examine the effects on hnRNP A1 binding to biotin-labeled EV71 5′UTR. Eluted proteins were analyzed by Western blot using anti-hnRNP A1 antibody (upper panel) or AUF1 antibody (lower panel). Lane 1: non-biotinylated RNA was added to the reaction as a negative control. Lanes 3–6: 0.2, 0.5, 1, and 1.5 µg of purified p40AUF1 was added to reactions, respectively. (B) The pull-down assay was performed as described in panel (A) except increasing amounts of purified recombinant FBP1 were added to the reaction mixtures. (C) Biotin-labeled SL-II was used in the pull-down assay to examine the effect of increasing amounts of p40AUF1 on hnRNP A1–SL-II interactions. (D) Biotin-labeled SL-II and increasing amounts of FBP1 were used in the pull-down assay to assess the effect of FBP1 on hnRNP A1–SL-II interactions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103827-g005: AUF1 can compete with hnRNP A1 for association with the IRES.(A) The pull-down assay was performed with SF268 lysate and biotin-labeled EV71 5′UTR as described for Figure 2. Additionally, increasing amounts of purified recombinant p40AUF1 were added to reaction mixtures to examine the effects on hnRNP A1 binding to biotin-labeled EV71 5′UTR. Eluted proteins were analyzed by Western blot using anti-hnRNP A1 antibody (upper panel) or AUF1 antibody (lower panel). Lane 1: non-biotinylated RNA was added to the reaction as a negative control. Lanes 3–6: 0.2, 0.5, 1, and 1.5 µg of purified p40AUF1 was added to reactions, respectively. (B) The pull-down assay was performed as described in panel (A) except increasing amounts of purified recombinant FBP1 were added to the reaction mixtures. (C) Biotin-labeled SL-II was used in the pull-down assay to examine the effect of increasing amounts of p40AUF1 on hnRNP A1–SL-II interactions. (D) Biotin-labeled SL-II and increasing amounts of FBP1 were used in the pull-down assay to assess the effect of FBP1 on hnRNP A1–SL-II interactions.
Mentions: One mechanism that might account for how AUF1 could reduce IRES activity is that it competes with positive-acting ITAFs for binding to the EV71 5′UTR, thereby reducing their access to the IRES. Our previous work demonstrated that hnRNP A1 associates with SL-II within the EV71 5′UTR and promotes viral translation and replication [16][43]. AUF1 also associates with SL-II (Fig. 2C). AUF1 and hnRNP A1 are homologous in amino acid sequence and functional domains, particularly in the two RRMs (RNA recognition motifs) and C-terminal glycine-rich region (see Supplementary Fig. 1 in [44]). To determine whether AUF1 and hnRNP A1 might compete for binding to the EV71 5′UTR, a protein-RNA pull-down and competition assay was performed. Biotinylated 5′UTR and 200 µg of protein from cell lysates were mixed in reactions that included increasing amounts of purified recombinant His6-p40AUF1. This isoform was chosen since it is predominantly cytoplasmic [45], and it is expressed in SF268 cells. Binding was measured by protein-RNA pull-down followed by Western blotting, similar to Figure 2. As shown in Figure 5A, the amount of hnRNP A1 that was RNA-associated decreased with increasing amounts of p40AUF1 added; binding by hnRNP A1 was reduced 73% with the maximum p40AUF1 added. As expected, increasing p40AUF1 in reactions led to its increased association with RNA. As a control for specificity, effects of added FBP1 on binding by hnRNP A1 were examined (Fig. 5B). The amount of hnRNP A1 pulled-down by biotin-5′UTR remained constant despite addition of increasing amounts of FBP1. This is because FBP1 binds the linker region (nt 637–745) of the 5′UTR, while hnRNP A1 binds SL-II [13]. To determine if AUF1 and hnRNP A1 compete for 5′UTR binding via SL-II, biotin-labeled SL-II was used in the RNA pull-down assay. As shown in Figure 5C, the amount of hnRNP A1 associated with SL-II decreased with increasing amounts of p40AUF1 added; binding by hnRNP A1 was decreased by 80% with the maximum amount of p40AUF1 added. By contrast, the amount of hnRNP A1 associated with SL-II remained constant with increasing amounts of FBP1 added; SL-II did not pull-down FBP1, since it does not bind SL-II, as noted above (Fig. 5D). Taken together, these results suggest reciprocal binding of SL-II by AUF1 and hnRNP A1.

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