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

Azlinda Anwar,K.M Leong,Mary L Ng,Justin J.H Chu,Mariano A Garcia-Blanco

doi:10.1074/jbc.m109.006239

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.

Highlights

dengue virus (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]
Huh-7 cells were infected at a multiplicity of infection (MOI) of 1, and the subcellular localization of DENV envelope (Env) and PTB was assessed 48 h post-infection (p.i.) by indirect immunofluorescence
We show that PTB is required for efficient dengue virus 2 New Guinea C strain (DENV-2) propagation in human cells in culture

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Viruses—The dengue virus 2 New Guinea C strain (DENV-2) and yellow fever virus 17D strain (YFV) used in this study were propagated in the C6/36 mosquito and Vero cells, respectively, as previously described [20]. RNA Immunoprecipitation (RIP)—Isolation of PTB-associated RNAs under native conditions was performed by immunoprecipitation as described previously [26] using the anti-PTB monoclonal antibody from precleared lysates of Huh-7 cells uninfected or infected with DENV-2 at an MOI of 1. The reporter transcripts were transfected into Huh-7 cells using Lipofectamine 2000 (Invitrogen) as previously described [28], together with a transfection control Renilla luciferase, obtained from in vitro transcription of a linearized pRL vector (Promega) The analyses of both firefly and Renilla luciferase levels were performed using the dual luciferase assay kit (Promega) on a Tecan Infinite M200 luminometer. Transfection and Generation of a Stable Cell Line Expressing DENV-2 NS4A-TAP Fusion Protein—DNA construct (pcTAPNS4A or pcTAP-control vector) were transfected into Huh-7 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. In 2% uranyl acetate for 5 min and dried before viewing under the EM208 transmission electron microscope (Philips)

RESULTS

DISCUSSION

Methods