During the replication of dengue virus, a viral non-structural glycoprotein, NS1, associates with the membrane on the cell surface and in the RNA replication complex. NS1 lacks a transmembrane domain, and the mechanism by which it associates with the membrane remains unclear. This study aimed to investigate whether membrane-bound NS1 is present in lipid rafts in dengue virus-infected cells. Double immunofluorescence staining of infected HEK-293T cells revealed that NS1 localized with raft-associated molecules, ganglioside GM1 and CD55, on the cell surface. In a flotation gradient centrifugation assay, a small proportion of NS1 in Triton X-100 cell lysate consistently co-fractionated with raft markers. Association of NS1 with lipid rafts was detected for all four dengue serotypes, as well as for Japanese encephalitis virus. Analysis of recombinant NS1 forms showed that glycosylated NS1 dimers stably expressed in HEK-293T cells without an additional C-terminal sequence, or with a heterologous transmembrane domain, failed to associate with lipid rafts. In contrast, glycosylphosphatidylinositol-linked recombinant NS1 exhibited a predilection for lipid rafts. These results indicate an association of a minor subpopulation of NS1 with lipid rafts during dengue virus infection and suggest that modification of NS1, possibly lipidation, is required for raft association. INTRODUCTIONDengue virus is an important mosquito-borne human pathogen causing illnesses ranging from mild febrile illness and dengue fever to life-threatening dengue haemorrhagic fever and dengue shock syndrome (Halstead, 1997). It consists of four serotypes and belongs to the genus Flavivirus of the family Flaviviridae, which includes viruses such as yellow fever virus, Japanese encephalitis virus (JEV), tick-borne encephalitis virus and West Nile virus (Lindenbach & Rice, 2001). The genome of flaviviruses is a single-stranded RNA that encodes at least 10 known proteins (C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5).NS1, a relatively conserved glycoprotein, exists in multiple forms in different compartments of virus-infected cells (Flamand et al., 1992;Jacobs et al., 2000;Leblois & Young, 1995;Mason, 1989). Following proteolytic cleavage of the viral polyprotein, NS1 is present in the lumenal side of the endoplasmic reticulum, mainly as homodimeric molecules (Falgout & Markoff, 1995). Intracellular dimeric NS1 associates with the cellular membrane, but the structural basis for membrane association is not known. NS1 lacks a transmembrane domain and there is no evidence for posttranslational protein modification that can explain its affinity for the membrane. The possibility of glycosylphosphatidylinositol (GPI) linkage as suggested by in vitro transfection and HeLa cell infection studies (Jacobs et al., Journal of General Virology (2008) Mackenzie et al., 1996;Muylaert et al., 1996Muylaert et al., , 1997. NS1 is also present on the cell surface and is released into the extracellular compartment. In the latter, NS1 is found in the form of hexamers ...

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BackgroundHost and viral proteins are involved in dengue virus (DENV) replication. Heterogeneous ribonucleoprotein (hnRNP) C1/C2 are abundant host cellular proteins that exhibit RNA binding activity and play important roles in the replication of positive-strand RNA viruses such as poliovirus and hepatitis C virus. hnRNP C1/C2 have previously been shown to interact with vimentin and viral NS1 in DENV-infected cells; however, their functional role in DENV replication is not clearly understood. In the present study, we investigated the role of hnRNP C1/C2 in DENV replication by using an in vitro model of DENV infection in a hepatocyte cell line (Huh7) and siRNA-mediated knockdown of hnRNP C1/C2.MethodsHuh7 cells were transfected with hnRNP C1/C2-specific siRNA or irrelevant siRNA (control) followed by infection with DENV. Mock and DENV-infected knockdown cells were processed for immunoprecipitation using hnRNP C1/C2-specific antibody or their isotype-matched control antibody. The immunoprecipitated samples were subjected to RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) for detection of DENV RNA. In addition, the knockdown cells harvested at varying time points after the infection were assessed for cell viability, cell proliferation, percentage of DENV infection, amount of viral RNA, and viral E and NS1 expression. Culture supernatants were subjected to focus forming unit assays to determine titers of infectious DENV. DENV luciferase reporter assay was also set up to determine viral translation.ResultsImmunoprecipitation with the anti-hnRNP C1/C2 antibody and subsequent RT-PCR revealed the presence of DENV RNA in the immunoprecipitated complex containing hnRNP C1/C2 proteins. Transfection with hnRNP C1/C2-specific siRNA resulted in a significant reduction of hnRNP C1/C2 mRNA and protein levels but did not induce cell death during DENV infection. The reduced hnRNP C1/C2 expression decreased the percentage of DENV antigen-positive cells as well as the amount of DENV RNA and the relative levels of DENV E and NS1 proteins; however, it had no direct effect on DENV translation. In addition, a significant reduction of DENV titers was observed in the supernatant from DENV-infected cells following the knockdown of hnRNP C1/C2.ConclusionsOur findings suggest that hnRNP C1/C2 is involved in DENV replication at the stage of viral RNA synthesis.

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Viruses manipulate the life cycle in host cells via the use of viral properties and host machineries. Development of antiviral peptides against dengue virus (DENV) infection has previously been concentrated on blocking the actions of viral structural proteins and enzymes in virus entry and viral RNA processing in host cells. In this study, we proposed DENV NS1, which is a multifunctional nonstructural protein indispensable for virus production, as a new target for inhibition of DENV infection by specific peptides. We performed biopanning assays using a phage-displayed peptide library and identified 11 different sequences of 12-mer peptides binding to DENV NS1. In silico analyses of peptide-protein interactions revealed 4 peptides most likely to bind to DENV NS1 at specific positions and their association was analysed by surface plasmon resonance. Treatment of Huh7 cells with these 4 peptides conjugated with N-terminal fluorescent tag and C-terminal cell penetrating tag at varying time-of-addition post-DENV infection could inhibit the production of DENV-2 in a time-and dosedependent manner. The inhibitory effects of the peptides were also observed in other virus serotypes (DENV-1 and DENV-4), but not in DENV-3. These findings indicate the potential application of peptides targeting DENV NS1 as antiviral agents against DENV infection. Dengue virus (DENV) infection is a major and increasing public health problem worldwide. There are approximately 390 million infections, 500,000 severe cases with hospitalization, and a 2.5% mortality rate each year 1,2. Although the majority of individuals experiencing DENV infection are asymptomatic, approximately one-fourth of the infected cases develop a wide range of clinical manifestations of dengue disease with unclear pathogenic mechanisms 3,4. To date, there is no specific anti-viral drug available for the treatment of DENV infection. A licensed dengue vaccine also has some limitations for use in naïve individuals not previously infected by DENV and in children less than 9 years of age 5,6. Consequently, the development of an alternative strategy to combat DENV infection and severe dengue is still needed. DENV is a positive, single-stranded, enveloped RNA virus belonging to the family Flaviviridae, and it has 4 distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) 7. Infection with any DENV serotype can generate

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Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.