Nuclear Localization of Dengue Virus Nonstructural Protein 5 Does Not Strictly Correlate with Efficient Viral RNA Replication and Inhibition of Type I Interferon Signaling

Abstract

dDengue virus (DENV) is an important human pathogen, especially in the tropical and subtropical parts of the world, causing considerable morbidity and mortality. DENV replication occurs in the cytoplasm; however, a high proportion of nonstructural protein 5 (NS5), containing methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities, accumulates in the nuclei of infected cells. The present study investigates the impact of nuclear localization of NS5 on its known functions, including viral RNA replication and subversion of the type I interferon response. By using a mutation analysis approach, we identified the most critical residues within the ␣␤ nuclear localization signal (␣␤NLS), which are essential for the nuclear accumulation of this protein. Although we observed an overall correlation between reduced nuclear accumulation of NS5 and impaired RNA replication, we identified one mutant with drastically reduced amounts of nuclear NS5 and virtually unaffected RNA replication, arguing that nuclear localization of NS5 does not correlate strictly with DENV replication, at least in cell culture. Because NS5 plays an important role in blocking interferon signaling via STAT-2 (signal transducer and activator of transcription 2) degradation, the abilities of the NLS mutants to block this pathway were investigated. All mutants were able to degrade STAT-2, with accordingly similar type I interferon resistance phenotypes. Since the NLS is contained within the RdRp domain, the MTase and RdRp activities of the mutants were determined by using recombinant full-length NS5. We found that the C-terminal region of the ␣␤NLS is a critical functional element of the RdRp domain required for polymerase activity. These results indicate that efficient DENV RNA replication requires only minimal, if any, nuclear NS5, and they identify the ␣␤NLS as a structural element required for proper RdRp activity. Dengue fever is the most common mosquito-borne viral disease affecting humans (1). An estimated 40% of the world's population lives in areas where dengue is endemic, and ϳ230 million infections occur annually worldwide (2, 3). With nearly 500,000 patients developing dengue hemorrhagic fever annually, leading to more than 20,000 deaths, dengue has emerged as a major public health problem with significant economic, political, and social impacts (1). However, neither antiviral drugs nor an approved vaccine is currently available (4, 5). The lack of a vaccine is due mainly to the existence of 4 different dengue virus (DENV) serotypes that can cause severe disease symptoms, especially upon secondary infection with a heterologous serotype.DENV is an enveloped, single-stranded RNA virus belonging to the genus Flavivirus in the family Flaviviridae (2). The DENV genome has a length of ϳ10,700 nucleotides and possesses a type I cap at its 5= end. The genome encodes a single polyprotein, which is cotranslationally and posttranslationally cleaved by host and viral proteases into 3 structural proteins (capsid [C], premembrane protein ...

Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization

“…For example, the Ebola virus VP24 protein binds to human KPNA1, -5, and -6, preventing the binding and subsequent nuclear translocation of phosphorylated STAT1, inhibiting IFN signaling (68,69), whereas the Hantaan virus N protein binds to KPNA1, -2, and -4, inhibiting the nuclear translocation and activity of NF-B (70). The results of our work and a recent study (46) suggest that nuclear localization of DENV NS5 is not strictly essential for DENV replication. However, previously, we found that mutations in DENV-2 NS5 that delay nuclear localization lead to decreased virus replication (30).…”

“…The mutation of the A2 cluster to Ala was found to delay NS5 nuclear localization. Studies investigating the effects of mutations made to DENV-2 NS5 a/bNLS on virus replication and POL activity, using bacterially expressed NS5, have shown that only the mutation R401A/K401A (Ser-401/ Lys-402 in DENV-4) abolished virus replication and POL activity (46,62), whereas other mutations could reduce viral replication without affecting POL activity (46), highlighting the pleiotropic effects of mutations in the a/bNLS region.…”

Characterization of the Mode of Action of a Potent Dengue Virus Capsid Inhibitor

“…Replication was determined by measuring luciferase activity in cell lysates 4, 24, 48, and 72 h after transfection. For harvest, cells were washed once with PBS and lysed by adding 200 l lysis buffer as previously described (19). Cells were frozen immediately at Ϫ70°C, and after thawing, lysates were resuspended by gentle pipetting.…”

“…The total volume of the reaction mixture was 15 l, and reactions were performed in two stages: stage 1, 15 min at 95°C; stage 2, 40 cycles of 15 s at 95°C and 60 s at 60°C. Quantities of DV RNA were calculated by using serial dilutions of known amounts of DV in vitro transcripts that were processed in parallel as previously described (19).…”

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

“…The activity of DENV-encoded Rluc was used as a measure for intracellular RNA replication of reporter replicons and viruses. Cells were lysed, and Rluc assays were performed exactly as previously described (33) by measuring luminescence using a Lumat LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany) upon addition of coelenterazine (1.43 M final concentration).…”