Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.is the causative agent of dengue fever, dengue hemorrhagic fever, and toxic shock syndrome (1). These diseases are prevalent in tropical regions around the world, where the mosquito vectors thrive. A total of 50 to 100 million DENV-related infections occur annually worldwide (2). Despite the large burden to human health, basic research into the development of DENV antiviral therapy has been limited.All positive-strand RNA viruses remodel cytosolic membranes to establish sites of replication (reviewed in ref.3). These structures play a critical role in the viral life cycle, likely by increasing the local concentration of replicating viral components and by sequestering viral antigens from recognition by host immune surveillance mechanisms. Following the initial translation and processing of DENV proteins, cellular membranes are remodeled to establish cytosolic replication complexes (RCs). DENV nonstructural protein 4A (NS4A) has been proposed to be sufficient for DENV membrane remodeling, perhaps performing a structural role by inducing membrane curvature (4). This function requires a processing event in which the C terminus of NS4A is removed by the viral NS2B-3 protease.Recently, the 3D structure of DENV RCs has been determined by electron tomography (5). That study clearly demonstrated that viral replication takes place on double-membrane vesicles that are contiguous with the endoplasmic reticulum (ER). Interestingly, there also appears to be physical linkage between sit...

Plasmodium falciparum causes the most severe form of malaria and kills up to 2.7 million people annually. Despite the global importance of P. falciparum, the vast majority of its proteins have not been characterized experimentally. Here we identify P. falciparum protein-protein interactions using a high-throughput version of the yeast two-hybrid assay that circumvents the difficulties in expressing P. falciparum proteins in Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid screens with P. falciparum protein fragments, we identified 2,846 unique interactions, most of which include at least one previously uncharacterized protein. Informatic analyses of network connectivity, coexpression of the genes encoding interacting fragments, and enrichment of specific protein domains or Gene Ontology annotations were used to identify groups of interacting proteins, including one implicated in chromatin modification, transcription, messenger RNA stability and ubiquitination, and another implicated in the invasion of host cells. These data constitute the first extensive description of the protein interaction network for this important human pathogen.

The single flagellum of the protozoan parasite Trypanosoma brucei is attached along the length of the cell body by a complex structure that requires the FLA1 protein. We show here that inhibition of FLA1 expression by RNA interference in procyclic trypanosomes causes flagellar detachment and prevents cytokinesis. Despite being unable to divide, these cells undergo mitosis and develop a multinucleated phenotype. The Trypanosoma cruzi FLA1 homolog, GP72, is unable to complement either the flagellar detachment or cytokinesis defects in procyclic T. brucei that have been depleted of FLA1 by RNA interference. Instead, GP72 itself caused flagellar detachment when expressed in T. brucei. In contrast to T. brucei cells depleted of FLA1, procyclic T. brucei expressing GP72 continued to divide despite having detached flagella, demonstrating that flagellar attachment is not absolutely necessary for cytokinesis. We have also identified a FLA1-related gene (FLA2) whose sequence is similar but not identical to FLA1. Inhibition of FLA1 and FLA2 expression in bloodstream T. brucei caused flagellar detachment and blocked cytokinesis but did not inhibit mitosis. These experiments demonstrate that the FLA proteins are essential and suggest that in procyclic T. brucei, the FLA1 protein has separable functions in flagellar attachment and cytokinesis.Trypanosoma brucei is an extracellular protozoan parasite that relies on a single flagellum for motility. This critical structure emerges from the flagellar pocket, a specialized secretory organelle near the posterior end of the cell, and extends along the cell body to the anterior tip. The flagellum contains an axoneme with the classical 9 ϩ 2 bundle of microtubules and a paraflagellar rod (PFR) 1 that is comprised primarily of two proteins, PFR-A and PFR-C (1, 2). The axoneme extends from the kinetoplast-linked basal body to the anterior tip of the flagellum. The PFR lies adjacent to the axoneme in the flagellum and is slightly shorter; it extends from the point where the flagellum exits the flagellar pocket to the tip. The PFR is required for motility; inhibition of PFR-A expression by RNA interference (RNAi) ablates the PFR and paralyzes procyclic trypanosomes (3).The flagellum is attached to the cell body via the flagellar attachment zone (FAZ), a complex but largely uncharacterized structure (4, 5). The FAZ is made up of an electron-dense cytoplasmic filament and a specialized set of four microtubules that are associated with the smooth endoplasmic reticulum (for a recent review of the T. brucei cytoskeleton, see Ref. 6). The filament is invariably located in a unique gap between two microtubules in the subpelicular cortex with the four microtubules always found immediately to the left when viewed from the posterior end. Cross-links extend from the filament across the cell and flagellum membranes and into the PFR.During cell division, the flagellum and FAZ must be duplicated and segregated to the daughter cells. Synthesis of the new flagellum begins with duplication of the basal ...