Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site

Abstract

Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5′UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5′UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM’s ability to bind RNA, abrogated the protein’s capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1′s ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein’s capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein’s ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.

The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK

“…The reporter activity of the first cistron, RLuc, was equivalent, indicating that equivalent mRNA amounts were transfected and that RLuc activity occurred in a cap-dependent fashion from all the reporters ( Figure 4 B). Similar to what has been previously described [ 38 , 52 , 53 ], the reporter activity of the second cistron, FLuc, was barely detected from the dl ∆EMCV reporter and was high when the HCV IRES was used as the intergenic region. Furthermore, we did not observe IRES activity when either of the 5′UTRs of SLE-1 were used as the intergenic regions ( Figure 4 C).…”

“…To test if the IRES activity observed in the in vitro assays could also be observed in cells, we delivered the bicistronic mRNA reporter into the HeLa cells. As the positive control of the IRES activity, we used the dl HCV IRES ( Figure 4 A) [ 40 , 52 , 53 ], containing the Hepatitis C IRES in the intergenic region, while our negative control was dl ∆EMCV ( Figure 4 A) that does not have an intergenic region and does not present IRES activity [ 37 , 40 , 54 , 55 ]. We also included the dl SLE-1 5′UTR S along our dl SLE-1 5′UTR AS as the control, since our in vitro experiments showed no translation activity.…”