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

Abstract

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 5Ј-and 3Ј-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 singlestranded 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 ...

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

“…However, at 48 h p.i., a discrete amount of PTB protein could be observed in the cytoplasm of infected Huh7 cells (Fig. 2), confirming previous observations (Anwar et al, 2009;Jiang et al, 2009). Furthermore, PTB present in the nucleus as well as the discrete amount of PTB present in the cytoplasm co-localized with DENV replicase, the nonstructural protein NS5 (Fig.…”

“…The massive relocation of PTB protein in Vero cells contrasts with the discrete movement reported for this protein in DENV-infected Huh7 cells (Anwar et al, 2009;Jiang et al, 2009). To confirm this difference, Huh7 cells were infected with DENV at an m.o.i.…”

“…In the present report, we have demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection in Vero cells. This translocation was only observed in Vero cells; in Huh7 cells, as described previously, only a partial translocation of PTB from the nucleus to the cytoplasm was detected (Anwar et al, 2009;Jiang et al, 2009), suggesting that some cell-type-specific factors present in Vero cells might be conditioning the massive translocation of PTB. At 48 h post-infection (p.i.…”

“…In other systems in which PTB participates in virus translation and/or replication, this protein has also been shown to move from the nucleus to the cytoplasm (Aizaki et al, 2006;Chang & Luo, 2006;Grover et al, 2008). However, in recent reports, Anwar et al (2009) andJiang et al (2009) observed only partial translocation of PTB from the nucleus to the cytoplasm of DENV-infected Huh7 cells. To verify the difference between the two cell types, an analysis of PTB location in Huh7 cells was performed.…”

Development of viable TAP-tagged dengue virus for investigation of host–virus interactions in viral replication

“…Interestingly, a previous study examining the infected Huh-7 proteome showed significant alterations in the abundance of RNA processing proteins (Pando-Robles et al, 2014). While DENV-2 NS5 could shuttle between the nucleus and cytoplasm, and thus hnRNPs might not be necessarily associated with the replication complex, it has been revealed that the hnRNPK and hnRNPA1 proteins accumulated in the cytoplasm and promoted DENV production (Anwar et al, 2009;Brunetti et al, 2015;Jiang et al, 2009). However, precise roles played by interaction of hnRNPs with NS5 require further study.…”