The Polypyrimidine Tract-Binding Protein Is a Transacting Factor for the Dengue Virus Internal Ribosome Entry Site

Abstract

Dengue virus (DENV) is an enveloped, positive sense, single-stranded RNA virus belonging to the Flaviviridae. Translation initiation of the DENV mRNA (vRNA) can occur following a cap-dependent, 5′-3’end-dependent internal ribosome entry site (IRES)-independent or IRES-dependent mechanism. This study evaluated the activity of DENV IRES in BHK-21 cells and the role of the polypyrimidine-tract binding protein (PTB) isoforms PTB1, PTB2, and PTB4 as IRES-transacting factors (ITAFs) for the DENV IRES. The results show that DENV-IRES activity is stimulated in DENV-replicating BHK-21 cells and cells expressing the Foot-and-mouth disease virus leader or Human rhinovirus 2A proteases. Protease activity was necessary, although a complete shutdown of cap-dependent translation initiation was not a requirement to stimulate DENV IRES activity. Regarding PTB, the results show that PTB1 > PTB2 > PTB4 stimulates DENV-IRES activity in BHK-21 cells. Mutations in the PTB RNA recognition motifs (RRMs), RRM1/RRM2 or RRM3/RRM4, differentially impact PTB1, PTB2, and PTB4’s ability to promote DENV IRES-mediated translation initiation in BHK-21 cells. PTB1-induced DENV-IRES stimulation is rescinded when RRM1/RRM2 or RRM3/RRM4 are disrupted. Mutations in RRM1/RRM2 or RRM3/RRM4 do not affect the ITAF activity of PTB2. Mutating RRM3/RRM4, but not RRM1/RRM2, abolishes the ability of PTB4 to stimulate the DENV IRES. Thus, PTB1, PTB2, and PTB4 are ITAFs for the DENV IRES.