Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

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Abstract

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.

Introduction

Dengue virus is a mosquito-borne human pathogen which causes a serious public health concern around the world with approximately 100 million cases of dengue infection and 500,000 cases of hospitalizations per annum [1]. The fatality rate of the affected individuals is about 1–5% and occurs mostly in children [1]. However, the mechanisms involved in the pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) remain unraveled.

Dengue virus is a positive, single-stranded RNA virus in the genus Flavivirus of the family Flaviviridae and contains a 11-kb genome encoding three-structural proteins (capsid, C; premembrane, prM; and envelope, E) and seven-nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [2]. In virus-infected cells, newly synthesized NS1 appears as a monomer in the lumen of the endoplasmic reticulum (ER) and subsequently undergoes glycosylation and dimerization as the protein is transported along the host secretory pathway to the cell surface and eventually to the extracellular milieu [3], [4], [5]. The exact roles of NS1 in each compartment are not clearly understood.

Secreted NS1 is found to activate complements in the presence or absence of specific antibodies and interact with human complement regulatory protein clusterin, potentially leading to viral and host immune complex formation and subsequent plasma leakage [6], [7]. The correlation between levels of secreted NS1 and disease severity has also been observed [7], [8]. Unlike the secreted form, cell surface-associated NS1 requires cross-linking of specific antibodies to induce efficient complement activation and intracellular signal transduction in response to dengue virus infection [3], [7].

How the NS1 molecule functions inside virus-infected cells is still elusive. A number of previous studies propose the role of intracellular NS1 in the maturation process of dengue virus [9], [10], [11]. The NS1 molecule co-localizes with double-stranded dengue viral RNA (dsRNA), and associates with intracellular membrane structures, which are presumed sites of virus replication, and possibly with other viral nonstructural proteins, including NS2A, NS3, NS4A, and NS5 to form viral replication complexes in virus-infected cells [9], [10], [11], [12]. Very little is known about the interplay between dengue virus NS1, host proteins, and cellular responses during dengue virus infection. We therefore hypothesized that the intracellular NS1 may interact with host cellular proteins to facilitate its proper folding, trafficking and/or to promote favorable environment for virus production in the host cell. Biochemical and proteomic approaches were utilized in this study to identify NS1-interacting proteins and subsequently confirm the protein–protein interaction.

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Materials and methods

Cell line, virus, and antibodies. A human embryonic kidney epithelial cell line, 293T, dengue virus serotype 2 strain 16681, and mouse monoclonal antibodies recognizing linear epitopes (1B2, NS1-1F, NS1-3F, and NS1-4F) or conformational epitopes (NS1-8.2 and 1A4) on dengue virus NS1 were obtained as described previously [13], [14], [15]. A mouse anti-human hnRNP C1/C2 monoclonal antibody (clone 4F4) was purchased from Santa Cruz Biotechnology, Inc., CA, USA.

Dengue virus infection and

Determination of a suitable condition for preparation of dengue virus-infected cell lysates

Initially, HEK 293T cell line that had been infected with dengue virus at an MOI of 1 were collected daily for 3 days and assessed for the percentage of dengue virus infection and cell viability. Mock-infected cells served as negative controls. Infection of HEK 293T cells with dengue virus for 48 h resulted in high levels of viral antigen expression (Fig. 1A) but low percentage of cell death (Fig. 1B). As a result, we employed this condition for preparing cell lysates and tested whether dengue

Acknowledgments

We thank Dr. Panisadee Avirutnan for technical assistance in HUVEC cultures. This work was supported by a research grant (BT-B-02-MG-B4-4801) from the National Center for Genetic Engineering and Biotechnology (to S.N.), Senior Research Scholar Grants from Thailand Research Fund (to P.M. and P.Y.), and Siriraj Graduate Thesis Scholarship from the Faculty of Medicine Siriraj Hospital, Mahidol University (to S.Se.).

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    These authors contributed equally to this work.

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