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J. Biol. Chem., Vol. 282, Issue 12, 8873-8882, March 23, 2007

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The Non-structural Protein 4A of Dengue Virus Is an Integral Membrane Protein Inducing Membrane Alterations in a 2K-regulated Manner^*

Sven Miller, Stefan Kastner, Jacomine Krijnse-Locker, Sandra Bühler¹, and Ralf Bartenschlager²

From the Department of Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany and the European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Dengue virus (DV) is a positive sense RNA virus replicating in the cytoplasm in membranous compartments that are induced by viral infection. The non-structural protein (NS) 4A is one of the least characterized DV proteins. It is highly hydrophobic with its C-terminal region (designated 2K fragment) serving as a signal sequence for the translocation of the adjacent NS4B into the endoplasmic reticulum (ER) lumen. In this report, we demonstrate that NS4A associates with membranes via 4 internal hydrophobic regions, which are all able to mediate membrane targeting of a cytosolic reporter protein. We also developed a model for the membrane topology of NS4A in which the N-terminal third of NS4A localizes to the cytoplasm, while the remaining part contains three transmembrane segments, with the C-terminal end localized in the ER lumen. Subcellular localization experiments in DV-infected cells revealed that NS4A resides primarily in ER-derived cytoplasmic dot-like structures that also contain dsRNA and other DV proteins, suggesting that NS4A is a component of the membrane-bound viral replication complex (RC). Interestingly, the individual expression of DV NS4A lacking the 2K fragment resulted in the induction of cytoplasmic membrane alterations resembling virus-induced structures, whereas expression of full-length NS4A does not induce comparable membrane alterations. Thus, proteolytic removal of the 2K peptide appears to be important for induction of membrane alterations that may harbor the viral RC. These results shed new light on the role of NS4A in the DV replication cycle and provide a model of how this protein induces membrane rearrangements and how this property may be regulated.

Received for publication, October 23, 2006 , and in revised form, February 2, 2007.

^* This work was supported in part by an unrestricted grant from the Bristol-Myers-Squibb Foundation and Grant DENCO; contract number 517708 from the EU. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: sandra_sparacio{at}med.uni-heidelberg.de. ² To whom correspondence may be addressed: Dept. of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany. Tel.: 49-6221-56-4569; Fax: 49-6221-56-4570; E-mail: ralf_bartenschlager{at}med.uni-heidelberg.de.

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