Functional interaction between cellular p100 and the dengue virus 3′ UTR

Host factors interacting with the dengue virus (DENV) 3' UTR are involved in virus replication, but their roles remain poorly understood. We used RNA affinity capture and mass spectrometry to identify p100 as a host cellular protein associated with the DENV 3' UTR. By using RNA immunoprecipitation and confocal immunofluorescence analysis we demonstrated an interaction between p100 and the 3' UTR in DEN V-infected cells. We identified the A4 region (the extensive stem loop structure at the 3' end) as the binding site of p100 by studying deletion mutants. p100 knockdown specifically reduced the levels of viral RNA and viral protein in DENV-infected cells. Furthermore, downregulation of p100 reduced the expression of a heterologously expressed luciferase-3' UTR(DENV) mRNA in an A4-dependent manner, confirming the binding data and the effects of p100 knockdown on viral replication. These results provide evidence that p100 interacts with the 3' UTR of DENV and is required for normal DENV replication.