Summary Autophagy influences numerous cellular processes, including innate and adaptive immunity against intracellular pathogens. However, some viruses, including dengue virus (DENV), usurp autophagy to enhance their replication. The mechanism for a positive role of autophagy in DENV infection is unclear. We present data that DENV induction of autophagy regulates cellular lipid metabolism. DENV infection leads to an autophagy-dependent processing of lipid droplets and triglycerides to release free fatty acids. This results in an increase in cellular β-oxidation, which generates ATP. These processes are required for efficient DENV replication. Importantly, exogenous fatty acids can supplant the requirement of autophagy in DENV replication. These results define a role for autophagy in DENV infection and provide a mechanism by which viruses can alter cellular lipid metabolism to promote their replication.

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...

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Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha colocalized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.antivirals ͉ membranous web ͉ PI4K-IIIa ͉ PIK4CA ͉ RNAi H CV depends on cellular membrane-trafficking pathways at each step of its life cycle, including clathrin-mediated endocytosis, lipid droplet formation for virion assembly, and lipoprotein secretory pathways for egress (1-3). Like all positivestranded RNA viruses, HCV reorganizes intracellular membranes to establish sites of viral replication (4). HCV proteins are synthesized first at the rough endoplasmic reticulum (ER). Then the nonstructural proteins form viral replication complexes. HCV replication is thought to be intimately associated with modified cellular membranes that have protease-and nucleaseresistant properties (5, 6). Expression of the entire HCV polyprotein or the nonstructural protein NS4B alone results in formation of a unique multi-vesiculated structure termed the ''membranous web'' thought to be derived, at least in part, from ER membrane (6-8). Studies show that nonstructural proteins and viral RNA synthesis are associated with membranous webs (7,9). For these reasons they are implicated as the sites of active viral replication, although the precise origin and composition of the web-associated vesicles remains unclear.Cytosolic membrane rearrangements are a con...

Coronavirus infection causes diffuse alveolar damage leading to acute respiratory distress syndrome. The absence of ex vivo models of human alveolar epithelium is hindering an understanding of COVID-19 pathogenesis. We report a feeder-free, scalable, chemically-defined, and modular alveolosphere culture system for propagation and differentiation of human alveolar type 2 cells/pneumocytes derived from primary lung tissue. Cultured pneumocytes express the SARS-CoV-2 receptor ACE2 and can be infected with virus. Transcriptome and histological analysis of infected alveolospheres mirrors features of COVID-19 lungs, including emergence of interferon mediated inflammatory responses, loss of surfactant proteins, and apoptosis. Treatment of alveolospheres with interferons recapitulates features of virus infection, including cell death. In contrast, alveolospheres pretreated with low dose IFNs show a reduction in viral replication, suggesting the prophylactic effectiveness of IFNs against SARS-CoV-2. Human stem cell-based alveolospheres thus provide novel insights into COVID-19 pathogenesis and can serve as a model for understanding human respiratory diseases.

Viruses have evolved complex and dynamic interactions with their host cell. In recent years, we have gained insight into the expanding roles for cellular lipids in the viral life cycle. In particular, viruses target lipid signaling, synthesis, and metabolism to remodel their host cells into an environment that is optimal for their replication. This review highlights examples from different viruses that illustrate the importance of these diverse virus-lipid interactions.

Influenza A virus is a major human pathogen responsible for seasonal epidemics as well as pandemic outbreaks. Due to the continuing burden on human health, the need for new tools to study influenza virus pathogenesis as well as to evaluate new therapeutics is paramount. We report the development of a stable, replication-competent luciferase reporter influenza A virus that can be used for in vivo imaging of viral replication. This imaging is noninvasive and allows for the longitudinal monitoring of infection in living animals. We used this tool to characterize novel monoclonal antibodies that bind the conserved stalk domain of the viral hemagglutinin of H1 and H5 subtypes and protect mice from lethal disease. The use of luciferase reporter influenza viruses allows for new mechanistic studies to expand our knowledge of virus-induced disease and provides a new quantitative method to evaluate future antiviral therapies.

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.