Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection.

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.

Peptides targeting dengue viral nonstructural protein 1 inhibit dengue virus production.

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 non-structural 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 dose-dependent 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.

Mass spectrometric analysis of host cell proteins interacting with dengue virus nonstructural protein 1 in dengue virus-infected HepG2 cells.

Dengue virus (DENV) infection is a leading cause of the mosquito-borne infectious diseases that affect humans worldwide. Virus-host interactions appear to play significant roles in DENV replication and the pathogenesis of DENV infection. Nonstructural protein 1 (NS1) of DENV is likely involved in these processes; however, its associations with host cell proteins in DENV infection remain unclear. In this study, we used a combination of techniques (immunoprecipitation, in-solution trypsin digestion, and LC-MS/MS) to identify the host cell proteins that interact with cell-associated NS1 in an in vitro model of DENV infection in the human hepatocyte HepG2 cell line. Thirty-six novel host cell proteins were identified as potential DENV NS1-interacting partners. A large number of these proteins had characteristic binding or catalytic activities, and were involved in cellular metabolism. Coimmunoprecipitation and colocalization assays confirmed the interactions of DENV NS1 and human NIMA-related kinase 2 (NEK2), thousand and one amino acid protein kinase 1 (TAO1), and component of oligomeric Golgi complex 1 (COG1) proteins in virus-infected cells. This study reports a novel set of DENV NS1-interacting host cell proteins in the HepG2 cell line and proposes possible roles for human NEK2, TAO1, and COG1 in DENV infection.

Association of dengue virus NS1 protein with lipid rafts.

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.

Characterization of dengue virus NS1 stably expressed in 293T cell lines.

Dengue virus NS1 is a viral nonstructural protein detected in sera of infected individuals and in infected cells. Multiple NS1 structural forms have been reported but the functional characteristics of these forms remain unknown. In this study, a set of 293T cell lines stably expressing recombinant dengue NS1 without additional C-terminal sequence (rNS1s), with a heterologous transmembrane segment (rNS1tm), or with the 26-residue N-terminal portion of NS2A (rNS1v1) was established to aid in the characterization of different NS1 forms. Each NS1 protein form had distinct phenotypes and the following properties were documented: (1) dissipated expression in the cytoplasm, dimerization, and N-glycosylation were observed, regardless of the forms of NS1 expressed; (2) the rNS1v1 and rNS1tm forms, but not the rNS1s, were observed prominently on the surface membrane; (3) only the rNS1v1 form incorporated ethanolamine, a precursor of the glycosylphosphatidylinositol moiety, and was partially sensitive to digestion with phosphatidylinositol-specific phospholipase C. The stable 239T transfectants expressing multiple forms of dengue NS1 may be a useful model to investigate the function of NS1 and the mechanism by which NS1 associates with membrane.