Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B

Dengue virus (DV) is a member of the family Flaviviridae. These positive strand RNA viruses encode a polyprotein that is processed in case of DV into 10 proteins. Although for most of these proteins distinct functions have been defined, this is less clear for the highly hydrophobic non-structural protein (NS) 4B. Despite its possible role as an antagonist of the interferon-induced antiviral response, this protein may play an additional more direct role for viral replication. In this study we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. We found that NS4B resides primarily in cytoplasmic foci originating from the endoplasmic reticulum. NS4B colocalizes with NS3 and double-stranded RNA, an intermediate of viral replication, arguing that NS4B is part of the membrane-bound viral replication complex. Biochemical analysis revealed that NS4B is an integral membrane protein, and that its preceding 2K signal sequence is not required for this integration. We identified three membrane-spanning segments in the COOH-terminal part of NS4B that are sufficient to target a cytosolic marker protein to intracellular membranes. Furthermore, we established a membrane topology model of NS4B in which the NH2-terminal part of the protein is localized in the endoplasmic reticulum lumen, whereas the COOH-terminal part is composed of three trans-membrane domains with the COOH-terminal tail localized in the cytoplasm. This topology model provides a good starting point for a detailed investigation of the function of NS4B in the DV life cycle. Dengue virus (DV) is a member of the family Flaviviridae. These positive strand RNA viruses encode a polyprotein that is processed in case of DV into 10 proteins. Although for most of these proteins distinct functions have been defined, this is less clear for the highly hydrophobic non-structural protein (NS) 4B. Despite its possible role as an antagonist of the interferon-induced antiviral response, this protein may play an additional more direct role for viral replication. In this study we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. We found that NS4B resides primarily in cytoplasmic foci originating from the endoplasmic reticulum. NS4B colocalizes with NS3 and double-stranded RNA, an intermediate of viral replication, arguing that NS4B is part of the membrane-bound viral replication complex. Biochemical analysis revealed that NS4B is an integral membrane protein, and that its preceding 2K signal sequence is not required for this integration. We identified three membrane-spanning segments in the COOH-terminal part of NS4B that are sufficient to target a cytosolic marker protein to intracellular membranes. Furthermore, we established a membrane topology model of NS4B in which the NH2-terminal part of the protein is localized in the endoplasmic reticulum lumen, whereas the COOH-terminal part is composed of three trans-membrane domains with the COOH-terminal tail localized in the cytoplasm. This topology model provides a good starting point for a detailed investigation of the function of NS4B in the DV life cycle. The mosquito-transmitted Dengue virus (DV) 3The abbreviations used are: DV, Dengue virus; aa, amino acids; CLIMP, cytoskeleton linking membrane protein; dsRNA, double-stranded RNA; eGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GalT, galactosyltransferase; GST, glutathione S-transferase; HCV, hepatitis C virus; HD, high density; IFN-α/β, interferon-α/β; KUNV, Kunjin virus; LD, low density; NGC, New Guinea C-strain; NS, non-structural; PBS, phosphate-buffered saline; pTMD, predicted trans-membrane domain; RC, replication complex; ER, endoplasmic reticulum; STAT, transducers and activators of transcription; TMD, trans-membrane domain; HA, hemagglutinin. is the causative agent of dengue fever, the most prevalent arthropod-borne viral illness in humans with 50-100 million individuals infected annually worldwide (1Gubler D.J. Trends Microbiol. 2002; 10: 100-103Abstract Full Text Full Text PDF PubMed Scopus (1159) Google Scholar). Dengue fever is characterized by high fever, chills, body aches, and skin rash and ranges from mild, flu-like symptoms to severe forms, which are dengue hemorrhagic fever and the dengue shock syndrome. The four serotypes of DV identified so far (DV1-4) belong to the genus Flavivirus in the family Flaviviridae. Flaviviruses are small, enveloped viruses that have a single-stranded genomic RNA of positive polarity ∼11 kb in length. This RNA serves as mRNA for translation of a large polyprotein (3,391 amino acids in case of DV2) at the rough endoplasmic reticulum. The viral polyprotein is co- and post-translationally processed into three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The amino termini of prM, E, NS1, and NS4B are generated upon cleavage by the host signal peptidase in the lumen of the ER, whereas processing of most of the other NS proteins and the COOH terminus of the C protein is carried out by the viral two-component protease NS2B-3 in the cytoplasm of DV-infected cells (2Amberg S.M. Nestorowicz A. McCourt D.W. Rice C.M. J. Virol. 1994; 68: 3794-3802Crossref PubMed Google Scholar, 3Cahour A. Falgout B. Lai C.J. J. Virol. 1992; 66: 1535-1542Crossref PubMed Google Scholar, 4Falgout B. Pethel M. Zhang Y.M. Lai C.J. J. Virol. 1991; 65: 2467-2475Crossref PubMed Google Scholar). For cleavage of the COOH terminus of NS1 an unknown ER resident protease seems to be responsible (5Falgout B. Markoff L. J. Virol. 1995; 69: 7232-7243Crossref PubMed Google Scholar). A Golgi-localized furin protease mediates cleavage of prM at a late state of infection to generate the M protein (6Murray J.M. Aaskov J.G. Wright P.J. J. Gen. Virol. 1993; 74: 175-182Crossref PubMed Scopus (69) Google Scholar, 7Stadler K. Allison S.L. Schalich J. Heinz F.X. J. Virol. 1997; 71: 8475-8481Crossref PubMed Google Scholar). Most, if not all NS proteins are involved in replication of the flavivirus RNA, which occurs in close association with cellular membranes in so called viral replication complexes (RCs) (8Mackenzie J.M. Khromykh A.A. Jones M.K. Westaway E.G. Virology. 1998; 245: 203-215Crossref PubMed Scopus (261) Google Scholar, 9Westaway E.G. Mackenzie J.M. Kenney M.T. Jones M.K. Khromykh A.A. J. Virol. 1997; 71: 6650-6661Crossref PubMed Google Scholar). NS5 is the RNA-dependant RNA polymerase and it carries in the NH2-terminal domain a methyltransferase important for the of the RNA L. B. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Rice C.M. Microbiol. PubMed Scopus Google Scholar, B. B. J. 2002; PubMed Scopus Google Scholar, J. Virology. PubMed Scopus Google Scholar). NS3 as the viral which the for A. McCourt D.W. Rice C.M. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In NS3 an RNA as as an RNA PubMed Scopus Google Scholar, Virology. 1993; PubMed Scopus Google Scholar). The NS1 a role in viral RNA replication, at an of viral RNA replication Rice C.M. J. Virol. 1997; 71: PubMed Google Scholar, Rice C.M. J. Virol. PubMed Google Scholar). is the functions of the hydrophobic proteins NS2A, NS4A, and NS4B. been that may to the viral to cellular membranes McCourt D.W. Rice C.M. Virology. PubMed Scopus Google Scholar). In these proteins to the of the host J. J. Virol. PubMed Scopus Google Scholar, Khromykh A.A. J. Virol. PubMed Scopus Google Scholar, M. A. A. PubMed Scopus Google Scholar). NS4B is the of the hydrophobic NS proteins of the In the case of DV, NS4B of amino acids and an of NS4B of the hepatitis C virus which sequence to the flavivirus NS4B is characterized to M. J. Virol. PubMed Scopus Google Scholar, M. Rice C.M. J. Virol. PubMed Scopus Google Scholar, M. Rice C.M. Virology. PubMed Scopus Google Scholar, M. M. A. J. Virol. PubMed Scopus Google this is not the case with flavivirus NS4B, and its membrane topology its role in the viral life are that the COOH-terminal part of the protein preceding NS4B a signal sequence that serves to NS4B into the lumen of the of the of this signal is called the 2K the 2K is the terminus of NS4B by the host in the ER The cleavage at the a NS2B-3 cleavage at the so called which is and NH2-terminal of the S.M. Rice C.M. J. Virol. 1993; PubMed Google Scholar). NS4B of the Kunjin virus to be the in and in the of cells E.G. Khromykh A.A. Kenney M.T. Mackenzie J.M. Jones M.K. Virology. 1997; PubMed Scopus Google Scholar). it not found in Westaway E.G. Virol. 1992; PubMed Scopus Google Scholar). and of the viral virus and J. Virol. PubMed Scopus Google Scholar, A.A. Westaway E.G. J. Virol. 74: PubMed Scopus Google an important role of this protein in viral RNA replication. NS4B of the DV, the and the fever virus identified as an of the of DV NS4B the signal by with and of that the amino acids of DV are sufficient for the of and that viral polyprotein processing is required for function M. J. L. M. A. J. Virol. PubMed Scopus Google Scholar). to a NS2A, to and the of the three proteins in of M. A. A. PubMed Scopus Google Scholar, M. J. L. M. A. J. Virol. PubMed Scopus Google Scholar). of DV NS4B, we characterized its subcellular localization, membrane association, and membrane topology in this We that NS4B is part of the viral replication and established a model of NS4B into cellular membranes. and of the K. K. J. Google and the green in with amino and the polymerase J. Virol. PubMed Scopus Google the to the For the infection of and cells the Guinea C by used at a of infection of The virus in and cells for at with the by and cells for used in and from and from of and from The used in to the with The used in a and a to the used for J. D.W. A Scholar). The of all determined by sequence and The of of as a for polymerase of DV For of for the amino acids of the DV proteins generated by and into the with the E, NS3, NS4B, and The for the amino acids of the protein with the and the and into the and of the The for the proteins NS3 NS4B and NS5 into the and of A of the as the for the of the NS4B to the enhanced green fluorescent protein the for by from the and into the by B. B. PubMed Scopus Google Scholar). with and an the The and used for the of for the NS4B to generate the The as the sequence for the DV protein by a with the sequence and an with the sequence which an and a The with and and into the with the of and and of used for the of of NS3, NS4B, and NS5 proteins. with the cells for at and and by in phosphate-buffered with protease by high and and proteins from the by with a the and with of and by and with proteins and from the by an as in the in New a of with the proteins For the by as of DV-infected cells to and to membranes. membranes with in and the and for at for at with a of the in and and to the cycle. with and by of the for at in with the DV and this 10 and as by the of the by with an used for to the of the infection at in with in PBS, with for in and for with for in which the cells as cells in with a and to a with into cells to the infected as infection cells by into by at for in and for 10 with of a and at for to and to to a of a and to in a at for from the to the of the of from by and as For membrane at for 10 to membranes and proteins. in PBS, for and in as a and proteins to at in and in with the in for at for 10 in in for with the in and as complexes the of the with a as and at for to The at for 10 to membranes and proteins. in and with for in the of by 10 a and the for to the by and and sequence with the and of E, NS3, NS4B, and the role of DV NS4B in the viral life we generated for E, NS3, NS4B, and DV proteins as proteins in and New with the The characterized by analysis of of DV-infected with proteins of the in DV-infected not in cells not These the of the that used this to not of of DV of DV NS4B by its subcellular in cells by at infection The of the determined by cells In these cells NS4B in the with a the ER in the most the of large NS4B foci the cytoplasm with in the a a of cytoplasmic foci In to E.G. Khromykh A.A. Kenney M.T. Mackenzie J.M. Jones M.K. Virology. 1997; PubMed Scopus Google we not NS4B in the of DV-infected at late The found in cells the that of DV NS4B to the used not a cytoplasmic and as for DV NS4B found for NS3 arguing that these proteins part of the DV replication complex. In NS5 found in the a at in the cytoplasm in with A. A. J. Gen. Virol. 1992; PubMed Scopus Google Scholar, M. Zhang L. M. J. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). the subcellular of DV NS4B in more we with cellular marker proteins in in protein resident in the and cytoskeleton linking membrane protein a marker for the of the rough the and as DV NS4B. In of NS4B with the marker protein the marker protein a marker protein for the intermediate the cytoplasmic foci to we of DV NS4B with these by DV NS4B and these we that NS4B at close to the ER membranes in DV-infected NS4B to the of RNA of the flavivirus positive strand RNA occurs in membrane-bound the of DV NS4B, we that this protein is part of the RNA replication this we of infected cells by an that double-stranded foci with this in DV-infected not in cells and in these foci with integral protein of the ER which a This subcellular from the in cells the DV NS4B we found an with the foci in the whereas NS4B foci in the not with NS3, and protease as as the structural protein with the foci in the whereas the in the not of and a the viral protein we found the E, NS3, and NS4B In and DV NS5 that primarily to the these that DV is found in from the ER and that NS4B as as NS3 and are in these that most the of viral RNA replication. NS4B an and 2K the intracellular membrane association of DV NS4B we to the of NS4B in of DV-infected to a and to from the of the and of by and by these proteins to low as by the of an integral ER membrane protein that used as a marker for the analysis DV NS4B in the its membrane association the of NS4B membrane association in more we its of with that and this the of DV-infected cells at and the membranes to high that proteins and membranes other membrane proteins M. Virology. PubMed Scopus Google and with high that proteins by into membrane J. PubMed Scopus Google Scholar). with used to membranes and to trans-membrane proteins. used as a for an integral membrane protein, in the with and in the high of the cellular membranes with In a large of the protein to the high high low the to NS4B we found that it to of with We that NS4B most is an integral trans-membrane that the 2K at the COOH terminus of fever virus serves as a signal sequence for the of NS4B into the lumen of the ER S.M. Rice C.M. J. Virol. 1993; PubMed Google Scholar). In of in fever virus NS4B may be of association with membranes in the of an NH2-terminal signal DV NS4B with into membranes in the of its preceding signal we membrane and by cells with COOH-terminal DV NS4B. with NS4B in infected all DV to with membrane of DV NS4B is from its preceding 2K signal NS4B in far that NS4B is an integral membrane protein and that membrane association not the 2K signal a membrane topology model of NS4B we of trans-membrane domains with by the sequence predicted in the COOH-terminal of NS4B at the by of whereas for the NH2-terminal of the protein more NH2-terminal by and and whereas is by in the NH2-terminal NS4B predicted by other a and cleavage at the occurs in the ER lumen by the host S.M. Rice C.M. J. Virol. 1993; PubMed Google the terminus of NS4B be in the ER lumen, at the COOH terminus of NS4B is generated by the viral protease in the B. Pethel M. Zhang Y.M. Lai C.J. J. Virol. 1991; 65: 2467-2475Crossref PubMed Google Scholar, Virology. 1991; PubMed Scopus Google and be at the cytoplasmic of the ER, at For this we that a topology with of NS4B and in its COOH-terminal is the most predicted and the are domains this the of NS4B membrane a model for the topology of NS4B a of the of NS4B at COOH terminus to the to the predicted by a to the of important for membrane into cells and cells and with an the cellular of by with the 2K to a cytoplasmic with the ER marker not A with the proteins in which the 2K signal sequence to the to the and to this found for the protein in which the preceding 2K sequence to as as for the proteins the to the proteins the the the and to not an These that and not and membrane of the of the membrane association of proteins in of in the whereas with the 2K of the protein to far the of and to the pTMD, found in the that these not sufficient to membrane association of In all in the of with These the of the and that and membrane association of these proteins in more by of membrane of cells with the with to analysis of the revealed that this the of proteins the in the with membranes not the that and are into intracellular membranes and as signal of NS4B. DV NS4B by the membrane topology of DV NS4B in this we cells with in which with COOH-terminal of NS4B of cells with and analysis with a these the of the part of the proteins cytoplasmic the of the preceding to as membrane In case of a be protease whereas cytoplasmic be in proteins the 2K the 2K with the to protease a of the of the membranes with the proteins the protein the 2K and and to this to in by of with revealed that the to a not we identified a in at of NS4B. The that the protein is and is that the sequence to the ER with all proteins this part of the protein most to the ER lumen of its sequence be out that a NS4B found in DV-infected cells not of of a protein we not the NS4B. in the of a of with the with of and with the proteins to and to These an of In the case of the protein to the COOH-terminal by the its cytoplasmic This that is the membrane of DV NS4B. In the case of the protein 2K and to a in This the protein and it the of that it not an of the the in we that the 2K and the NH2-terminal of NS4B, whereas of NS4B to This provides for the of a in the to of DV NS4B and it the of a cytoplasmic preceding this analysis of of to cells revealed that most of the protein protease the cytoplasmic of This is in with the that the and of NS4B carries a a of not The of this that the amino acids of NS4B and the COOH-terminal This and it is possible that a of the COOH terminus of NS4B resides in the ER lumen, whereas the is the cytoplasmic the that is not by most to a these that the 2K and the membrane from the cytoplasmic to the and and from the to the cytoplasmic and most not the to in the ER The COOH terminus of NS4B is the cytoplasmic may the membrane with low In this we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. of NS4B in of cells we found that NS4B is localized in and large cytoplasmic foci viral replication. revealed that these to from the ER, and most not from the the from the intermediate viral proteins and NS3, and viral dsRNA, an intermediate of viral replication, found in these arguing that NS4B is part of the membrane-bound the RNA polymerase of the found in the of infected cells in with A. A. J. Gen. Virol. 1992; PubMed Scopus Google Scholar, M. Zhang L. M. J. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The of are in part with the of and M. J. L. M. A. J. Virol. PubMed Scopus Google Scholar). found that NS4B colocalizes with the ER marker in cells with a for DV In to not cytoplasmic foci a arguing that cells with this not the in infected it is the cytoplasmic foci are and foci have been identified in cells infected with other positive RNA for the Westaway E.G. Virol. PubMed Scopus Google Scholar). This virus membrane called membranes and the of viral polyprotein In ER and of in as small, membrane that to the viral E.G. Mackenzie J.M. Kenney M.T. Jones M.K. Khromykh A.A. J. Virol. 1997; 71: 6650-6661Crossref PubMed Google Scholar, Westaway E.G. Virol. 1992; PubMed Scopus Google Scholar, Westaway E.G. J. Virol. 1992; PubMed Scopus Google Scholar, K. J. PubMed Scopus Google Scholar, J.M. Jones M.K. Virology. PubMed Scopus Google Scholar, Virol. PubMed Scopus Google Scholar). is possible that the cytoplasmic foci in DV of these membrane The in the ER from the to in DV-infected cells and the of with NS3, NS4B, and in these foci this Although are that NS4B of Flaviviruses is important for RNA replication J. Virol. PubMed Scopus Google Scholar, A.A. Westaway E.G. J. Virol. 74: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Virology. PubMed Scopus Google NS4B not in in to NS1, NS2A, NS3, and Westaway E.G. Virol. 1992; PubMed Scopus Google Scholar). NS4B not into the of polyprotein in to NS2B, NS3, and the of DV NS4B with as in study a role of this protein in DV replication. are in to the role of the DV NS4B protein in the viral life cycle. that NS4B of is to distinct membrane in cells B. L. K. J. Virol. 2002; PubMed Scopus Google Scholar). We and not of and cells with for DV NS4B NS4B E.G. Khromykh A.A. Kenney M.T. Mackenzie J.M. Jones M.K. Virology. 1997; PubMed Scopus Google Scholar, M. J. L. M. A. J. Virol. PubMed Scopus Google Scholar). These that the NS4B proteins of of the family out and of DV that NS4B proteins of are to the ER membrane by the 2K signal sequence in the preceding protein S.M. Rice C.M. J. Virol. 1993; PubMed Google Scholar). We found that DV NS4B with intracellular membranes. by we that this protein is into membranes from the signal sequence in that NS4B signal These are with the of in translation of fever virus NS4B. that of this protein be of into association with membranes in the of an NH2-terminal signal sequence S.M. Rice C.M. J. Virol. 1993; PubMed Google Scholar). the membrane association and the topology of DV NS4B in more we established a model from from other by predicted in the COOH-terminal of NS4B by all whereas the NH2-terminal of the protein more to The three COOH-terminal be by are amino acids and of NS4B, and are sufficient to target eGFP, a cytosolic protein, to intracellular membrane This that as signal of DV NS4B. predicted by three of in the NH2-terminal of the protein and not sufficient to membrane of and to the model in The 2K and the membrane from the cytoplasmic to the and and from the to the cytoplasmic The and most not the The terminus of DV NS4B is localized in the ER lumen it is processed by the of the host The COOH terminus resides in the cytoplasm cleavage by the viral protease We that the topology of NS4B its from the topology of NS4B in the of the viral to NS4B topology by DV-infected cells not the of into of NS4B to a of viral RNA replication. and DV is the cytoplasmic and other NS4B are not is not possible at this to NS4B topology in DV-infected it is to the topology of NS4B be it is out of the of the it is possible that the of the COOH terminus of a of proteins is an of the sequence at the COOH the other if this topology for NS4B, that the COOH-terminal in a of NS4B post-translationally the membrane cleavage by the viral of membrane proteins have been for other viral for for the M protein of the virus J. L. J. Virol. PubMed Scopus Google Scholar, J.M. L. J. Virol. 1995; 69: PubMed Google the hepatitis virus large protein J. 1994; PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1994; PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google and the terminus of the NS4B M. M. A. J. Virol. PubMed Scopus Google Scholar). The function of flavivirus NS4B is its to the signal by with of M. A. A. PubMed Scopus Google Scholar, M. J. L. M. A. J. Virol. PubMed Scopus Google Scholar). have that of NS4B are required for M. J. L. M. A. J. Virol. PubMed Scopus Google Scholar). and M. J. L. M. A. J. Virol. PubMed Scopus Google that this part of the protein is localized in the cytoplasm the and the of NS4B. the topology model established for NS4B and for the topology of NS4B of other of the family M. M. A. J. Virol. PubMed Scopus Google Scholar, L. Rice C.M. J. Virol. PubMed Scopus Google Scholar). that the DV NS4B responsible for seems to be in the ER lumen For this a direct DV NS4B and cellular cytoplasmic involved in is not We that of is an for by of cellular of the from this function that flavivirus NS4B an additional more direct role for RNA replication. analysis that NS4B be involved in the replication in the and the host Virology. PubMed Scopus Google Scholar). Furthermore, and with viral virus and an important role of NS4B in viral replication J. Virol. PubMed Scopus Google Scholar, A.A. Westaway E.G. J. Virol. 74: PubMed Scopus Google Scholar). in the case of NS4B, which sequence of DV NS4B a an ER membrane found M. Rice C.M. Virology. PubMed Scopus Google Scholar, M. M. A. J. Virol. PubMed Scopus Google Scholar, M. Mackenzie J.M. Westaway E.G. J. Virol. PubMed Scopus Google Scholar, K. M. M. J. Gen. Virol. 1993; 74: PubMed Scopus Google Scholar). association of NS4B occurs and the protein an integral membrane protein M. Rice C.M. Virology. PubMed Scopus Google Scholar). A topology model and the topology model for the NS4B M. M. A. J. Virol. PubMed Scopus Google revealed that the COOH-terminal of NS4B whereas the NH2-terminal part may an M. Rice C.M. J. Virol. PubMed Scopus Google Scholar). functions have been for NS4B of HCV, translation M. 2002; PubMed Scopus Google Scholar, J. M. J. Virol. 2002; 66: PubMed Scopus Google of A. M. B. J. 2002; Full Text Full Text PDF PubMed Scopus Google and of cells J.M. M.K. PubMed Scopus Google Scholar). In it that NS4B intracellular membrane involved in the of the that is the of replication B. L. K. J. Virol. 2002; PubMed Scopus Google Scholar). In the function of DV NS4B to be In a of NS4B. topology model may be an starting point for a detailed investigation of the functions of this protein in the viral life cycle. We for and for the of this We and for the We are to for the and for the We the of at the of for to with