Elsevier

Virus Research

Volume 102, Issue 2, 15 June 2004, Pages 151-163
Virus Research

The non-structural 3 (NS3) protein of dengue virus type 2 interacts with human nuclear receptor binding protein and is associated with alterations in membrane structure

https://doi.org/10.1016/j.virusres.2004.01.025Get rights and content

Abstract

Flaviviral infections produce a distinct array of virus-induced intracellular membrane alterations that are associated with the flaviviral replication machinery. Currently, it is still unknown which flaviviral protein(s) is/are responsible for this induction. Using yeast two-hybrid and co-immunoprecipitation analyses, we demonstrated that the NS3 protein of dengue virus type 2 interacted specifically with nuclear receptor binding protein (NRBP), a host cellular protein that influences trafficking between the endoplasmic reticulum (ER) and Golgi, and that interacts with Rac3, a member of the Rho-GTPase family. Co-expression of NS3 and NRBP in baby hamster kidney cells exhibited significant subcellular co-localization, and revealed the redistribution of NRBP from the cytoplasm to the perinuclear region. Furthermore, a set of membrane structures affiliated with the rough ER at the perinuclear region was induced in cells transfected with NS3. These structures are reminiscent of the virus-induced convoluted membranes previously observed in flavivirus-infected cells. This interaction between dengue viral and host cell proteins as well as the formation of the NS3-induced membrane structures suggest that NS3 may subvert the role of NRBP in ER-Golgi trafficking.

Introduction

Dengue viruses are members of the genus Flavivirus which comprises over 70 members separated into groups on the basis of molecular phylogenetic analyses and serological relatedness (Kuno et al., 1998). Other members of this genus include Japanese encephalitis (JE), West Nile (WN), yellow fever (YF) and tick-borne encephalitis viruses. Infections caused by the four serotypes of dengue viruses borne by the Aedes mosquito can result in dengue fever (DF), dengue hemorrhagic fever (DHF) and/or dengue shock syndrome (DSS).

During virus replication, the single positive-sense viral genomic RNA is translated as a polyprotein in the order 5′-C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′. The first three proteins are viral structural proteins whereas the remaining proteins are viral non-structural (NS) proteins. The polyprotein is co- and post-translationally processed by cellular and viral proteases to produce the individual viral proteins (Chambers et al., 1990).

Hypertrophic proliferation of intracellular membranes is a prominent feature of flavivirus infections (Rice, 1996). The rough endoplasmic reticulum (ER) of infected cells proliferates and becomes distended during the early hours of infection. This is followed by the appearance of smooth membrane vesicles (SMVs) containing “thread-like” structures of varying sizes (60–80 nm) near the perinuclear region within the distended lumen of the ER. In most flavivirus infections, nucleocapsids and morphologically mature virions are first observed to accumulate in the lumen of the ER (Ng and Hong, 1989, Ng et al., 1994, Wang et al., 1997, Barth, 1999, Matthews et al., 2000). An exception is the Sarafend strain of WN virus whose virions bud at the plasma membrane (Ng et al., 2001). Paracrystalline (PC) arrays of viral particles are also observed in cells infected with St. Louis encephalitis and Kunjin viruses (Hong and Ng, 1987, Ng and Hong, 1989, Ng et al., 1994, Rice, 1996). At late hours of infection, convoluted membranes (CMs) are found amongst PC arrays, and may represent an interconversion of the PC arrays to CMs. The rough ER, SMVs and PC arrays appear to be interconnected. The elongated rough ER is often continuous with the distended rough ER lumen containing the SMVs, and the SMVs are consistently adjacent to the PC arrays (Ng and Hong, 1989, Wang et al., 1997). The proliferated rough ER, SMVs, CMs and PC arrays are proposed to constitute the viral replication factories (Mackenzie and Westaway, 2001). The “thread-like” structures within the SMVs that disappear at late stages of viral infection concomitant with the appearance of CMs are postulated to be progeny viral RNA (Ng, 1987, Ng and Hong, 1989, Wang et al., 1997, Matthews et al., 2000).

Using immunolocalization techniques, viral dsRNA (the replicative form), NS1 and NS3 are found to co-localize in membrane structures identified as vesicle packets synonymous with SMVs (Mackenzie et al., 1996, Westaway et al., 1997, Barth, 1999). The viral protease NS2B-NS3 co-localizes to PC arrays and CMs. These structures are not labeled by anti-NS1 nor anti-dsRNA antibodies that are associated with sites of RNA synthesis (Westaway et al., 1997, Westaway et al., 1999). In contrast, labeling of the E and prM proteins is found on reticulum membranes continuous with CMs and PC arrays, and on membranes at the periphery of CMs and PC arrays (Mackenzie and Westaway, 2001). The PC and CM structures are thus proposed to be sites of proteolytic processing of nascent viral polyprotein by the viral protease NS2B-NS3. The E and NS3 proteins also localize to the SMVs in JE virus-infected cells (Wang et al., 1998). Accumulating evidence strongly indicates that viral-induced membrane structures are the sites of synthesis of progeny viral RNAs (vesicle packets or SMVs). Proteolytic processing of the nascent viral polyprotein (PC arrays and CMs) and the assembly of virions (rough ER) are observed to occur at these sites.

The second largest flaviviral protein, NS3 (68–70 kDa), is also the second most highly conserved viral protein among flaviviruses. The NS3 protein does not contain long stretches of hydrophobic amino acids but becomes membrane-associated. This association probably occurs via interaction of NS3 with the hydrophobic NS2B protein, which together constitute the functional viral protease, NS2B-NS3 (Rice, 1996, Leung et al., 2001). The N-terminal 180 amino acids of NS3 contain the catalytic domain of the viral protease, as defined by sequence alignment with known serine proteases of the trypsin superfamily, by deletion analyses, and by site-directed mutagenesis of residues in the putative catalytic triad or the substrate-binding pocket. The residues of the catalytic triad, His-Asp-Ser, are conserved among flaviviruses (Chow et al., 1993, Ryan et al., 1998). The NS2B-NS3 protease is required for processing and cleavage of the viral polyprotein at the virC/anchC, NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/NS4B and NS4B/NS5 sites, and within the NS2A and NS3 regions (Rice, 1996). Additional cleavage sites occur within NS2A and NS3. The cleavage sites for the NS2B-NS3 protease usually consist of two basic residues followed by an amino acid with a short side chain (Arg/Lys/Gln)–(Arg/Lys)↓(Gly/Ser/Ala/Thr) (Chambers et al., 1990).

In addition, the C-terminus of NS3 contains significant regions of homology to the D–E–A–D family of RNA helicases (Chow et al., 1993, Kadare and Haenni, 1997), which are also present in homologous proteins of other positive-strand RNA viruses and in proteins with nucleoside triphosphatase (NTPase) binding motifs. The RNA-stimulated NTPase activity has been demonstrated using full-length and N-terminally truncated NS3 protein from WN, YF and JE viruses. Viral NTPase and RNA helicase activities have been reported for dengue virus type 2 NS3 protein (Li et al., 1999). NS3 and the flaviviral RNA-dependent RNA polymerase (RDRP), NS5, exist as a stable complex in cells infected with dengue virus type 2 (Kapoor et al., 1995). NS3 also co-immunoprecipitates with NS5 (Westaway et al., 1997), and with membrane fractions containing RDRP activity (Chu and Westaway, 1992). Interactions between NS3 and NS5 were also confirmed by yeast two-hybrid analysis (Johansson et al., 2001). These observations are consistent with the proposed role of NS3 in the function of the flaviviral replication complex. The NS3 protein is involved in flavivirus envelope formation (Yamshchikov and Compans, 1995) and in virus assembly (Liu et al., 2002). Mutations in NS3 helicase domain abolish viral replication (Matusan et al., 2001).

In this study, we employed the yeast-two hybrid system to identify putative cellular interacting partners of dengue virus NS3 protein. We found that NS3 interacts with nuclear receptor binding protein (NRBP), a polypeptide implicated in ER-Golgi trafficking. This interaction appeared to affect the intracellular distribution of NRBP, and may be important in the generation of virus-induced membrane structures and viral maturation during dengue virus replication in infected cells.

Section snippets

Dengue virus strain and cell lines

Dengue virus type 2 (New Guinea C strain) was propagated in the C6/36 mosquito cell line in L15 medium (GIBCO) containing 2% fetal calf serum (FCS). Baby hamster kidney (BHK-21 clone 13) cells were cultured in RPMI 1640 medium containing 10% FCS. COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS.

Construction of expression plasmids

Viral RNA was isolated from dengue virus-infected cell culture supernatant using Trizol LS reagent (Invitrogen), and converted to cDNA using primer D2RN and

Dengue virus NS3 interacts with host cellular NRBP

To identify cellular proteins that interact with the dengue NS3 protein, the full-length NS3 protein (inclusive of the N-terminal seven amino acids of the adjacent NS4A protein) was fused to the GAL4 DNA-binding domain to serve as bait in the yeast two-hybrid screen against a human bone marrow cDNA library. The latter was selected since macrophages, derived from hematopoietic myeloid stem cells, are reported to be the primary site of dengue virus infection (Bhamarapravati, 1997).

The initial

Discussion

Using yeast two-hybrid, co-immunoprecipitation and immunofluorescence techniques, we have demonstrated that dengue NS3 protein is able to interact with nuclear receptor binding protein (NRBP), a host cellular protein previously identified as an adaptor protein containing a kinase homology domain. The NRBP gene is ubiquitously expressed, with strong expression in testis, placenta, skeletal muscle, heart, ovary, leukocyte, brain, pancreas, spleen, prostate, kidney, intestine, liver and lung (

Acknowledgements

This study was supported by grants from the National Medical Research Council and Biomedical Research Council, Singapore. J.J.E. Chua was a recipient of a Research Scholarship from the National University of Singapore.

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