The Polypyrimidine Tract-binding Protein Is Required for Efficient Dengue Virus Propagation and Associates with the Viral Replication Machinery

The polypyrimidine tract-binding protein (PTB) functions primarily as an IRES trans-acting factor in the propagation of hepatitis C virus and picornaviruses. PTB interacts with secondary structures within the 3′- and 5′-untranslated regions of these viral genomes to mediate efficient IRES-mediated viral translation. PTB has also been reported to bind to the untranslated region of the single-stranded RNA dengue virus (DENV), suggesting a similar function for PTB in flaviviruses. Indeed small interfering RNA-mediated PTB knockdown inhibited the production of infectious DENV, and this inhibition was specific to PTB knockdown and not due to a nonspecific anti-viral state. In fact, PTB depletion did not inhibit the production infectious yellow fever virus, another flavivirus. Nevertheless, whereas PTB knockdown led to a significant decrease in the accumulation of DENV viral RNAs, it did not impair translation. Moreover, PTB was shown to interact with the DENV nonstructural 4A protein, a known component of the viral replication complex, and with the DENV genome during infection. These data suggest that PTB interacts with the replication complex of DENV and is acting at the level of viral RNA replication.Dengue virus (DENV)³ is the etiologic agent of dengue fever, currently the most prevalent arthropod-borne viral disease of humans (1). Dengue fever can be caused by any of four closely related but antigenically distinct DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). DENV belongs to the Flaviviridae family, which comprises other medically important pathogens including the Japanese encephalitis, yellow fever (YFV), and hepatitis C (HCV) viruses (2).The Flaviviridae family of viruses has single-stranded positive polarity RNA genomes, which are mRNAs coding viral proteins required for replication. The viral RNA-dependent RNA polymerase, NS5, in conjunction with other viral nonstructural (NS) proteins and host cellular proteins, copies complementary negative strand RNAs from the genomic RNA template, which in turn serve as templates for the synthesis of new positive strand RNAs (2). Replication, transcription, and assembly of virions require cis-acting elements within the 5′- and 3′-UTRs of the viral genomic RNA (3–6). These elements interact with host cellular RNA-binding proteins including the polypyrimidine tract-binding protein (PTB). PTB, a heterogeneous nuclear ribonucleoprotein (7), is involved in multiple aspects of cellular mRNA metabolism, including the regulation of alternative splicing, RNA stability, and internal ribosomal entry (IRES)-mediated translation of viral and cellular mRNAs (8–10).The role of PTB in the propagation of the positive single-stranded RNA viruses has largely been studied with HCV and picornaviruses, for which PTB primarily functions as an IRES trans-acting factor (ITAF), activating viral translation initiation (11, 12). Its role in RNA replication of these viruses is however still debated (13–16). PTB has also been reported to bind to the UTR of the DENV-4 (17) and Japanese encephalitis virus (18) flaviviruses. Flaviviruses are not known to contain an IRES within their RNA genomes (19), and thus the functional relevance of PTB in flavivirus propagation remains to be demonstrated. This study reports that RNA interference-mediated PTB depletion abrogated DENV production and reduced viral RNA levels. The interactions of PTB with the DENV genome and nonstructural 4A (NS4A) protein, components of the viral replication machinery, were observed during infection. Depletion of PTB and its paralog neuronal PTB (nPTB) did not alter translation from the DENV RNA. The requirement of PTB for flavivirus replication in general was explored using PTB- and nPTB-depleted cells for the propagation of YFV, with no significant inhibition of propagation observed. These data indicate a critical role for PTB in DENV and suggest an effect on RNA accumulation.