Membrane Trafficking Genes Research Articles

ER-Golgi-localized proteins TMED2 and TMED10 control the formation of plasma membrane lipid nanodomains.

To promote infections, pathogens exploit host cell machineries such as structural elements of the plasma membrane. Studying these interactions and identifying molecular players are ideal for gaining insights into the fundamental biology of the host cell. Here, we used the anthrax toxin to screen a library of 1,500 regulatory, cell-surface, and membrane trafficking genes for their involvement in the intoxication process. We found that endoplasmic reticulum (ER)-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the plasma membrane of human cells, an essential step dependent on localization to cholesterol-rich lipid nanodomains. Biochemical, morphological, and mechanistic analyses showed that TMED2 and TMED10 are essential components of a supercomplex that operates the exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.

Network and Evolutionary Analysis Reveals Candidate Genes of Membrane Trafficking Involved in Maize Seed Development and Immune Response

The plant membrane-trafficking system plays a crucial role in maintaining proper cellular functions and responding to various developmental and environmental cues. Thus far, our knowledge of the maize membrane-trafficking system is still limited. In this study, we systematically identified 479 membrane-trafficking genes from the maize genome using orthology search and studied their functions by integrating transcriptome and evolution analyses. These genes encode the components of coated vesicles, AP complexes, autophagy, ESCRTs, retromers, Rab GTPases, tethering factors, and SNAREs. The maize genes exhibited diverse but coordinated expression patterns, with 249 genes showing elevated expression in reproductive tissues. Further WGCNA analysis revealed that five COPII components and four Rab GTPases had high connectivity with protein biosynthesis during endosperm development and that eight components of autophagy, ESCRT, Rab, and SNARE were strongly co-upregulated with defense-related genes and/or with secondary metabolic processes to confer basal resistance to Fusarium graminearum. In addition, we identified 39 membrane-trafficking genes with strong selection signals during maize domestication and/or improvement. Among them, ZmSec23a and ZmVPS37A were selected for kernel oil production during improvement and pathogen resistance during domestication, respectively. In summary, these findings will provide important hints for future appreciation of the functions of membrane-trafficking genes in maize.

Alternative splicing regulation of membrane trafficking genes during myogenesis.

Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.

The Vesicle‐Mediated Transport Genes, Snap23 , Tmed2 , and Trip10 , are Alternatively Spliced During Striated Muscle Development

The reprogramming of alternative splicing patterns during development is a hallmark of tissue maturation and identity. Heart and skeletal (striated) muscle tissues exhibit the most highly conserved splicing programs, which substantially change during postnatal development. Notably, alterations of these splicing events are associated with severe cardiac and muscular diseases. In striated muscle, a collection of genes encoding membrane trafficking proteins are spliced in a developmental stage-specific manner. However, few splice isoforms have been characterized, and little is known about their functional roles during heart and skeletal muscle development. We hypothesize that coordinated splicing of membrane trafficking genes represents a splicing network, which functions to regulate the transport of cargo that is critical for muscle development and homeostasis. Here, we characterize three proteins involved in vesicle-mediated transport that are alternatively spliced in striated muscle: SNAP23, TMED2, and TRIP10. The synaptosome-associated protein 23 (SNAP23) is a SNARE protein that mediates vesicle fusion with the plasma membrane during exocytosis, the transmembrane emp23 domain-containing protein 2 (TMED2) regulates vesicle budding during secretion, and the thyroid hormone receptor interactor 10 (TRIP10) is involved in endocytosis and membrane tubulation. First, we found that during striated muscle development, the Snap23, Tmed2, and Trip10 pre-mRNAs undergo splicing changes of a single exon. Second, these three splicing transitions are characterized by a shift from short to long isoform expression that are regulated by the RBPs quaking (QKI) and polypyrimidine tract binding protein 1 (PTBP1). Third, depletion of SNAP23 in muscle cells results in reduced cell viability and differentiation, which can be rescued with conditioned media from differentiated cells or by seeding cells on collagen-coated plates. Finally, exclusive expression of the short SNAP23 isoform leads to increased fusion of myoblasts into myotubes that exhibit greater myotube area and a higher nuclei-per-myotube ratio. Taken together, our data suggest that splicing of Snap23 is an important regulator of myogenesis, where SNAP23 isoform expression controls secretion of cargo that is necessary for muscle cell fusion and myotube size. Preliminary studies show that disruption of TMED2 and TRIP10 isoform levels in muscle cells likewise affects differentiation. Elucidating the mechanisms of regulation and the functional consequences of splicing networks will be fundamental to our understanding of muscle development and disease.

The transcriptional landscape of Venezuelan equine encephalitis virus (TC-83) infection.

Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole.

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high‐throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.

A CRISPR Screen Using Subtilase Cytotoxin Identifies SLC39A9 as a Glycan-Regulating Factor

SummarySubtilase cytotoxin (SubAB) is a virulence factor produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains. The toxin recognizes sialoglycans for entry and cleaves an endoplasmic reticulum chaperon, binding immunoglobulin protein, to cause cell death. However, no systematic screening has yet been performed to identify critical host factors. Here, we performed a genome-wide CRISPR/Cas9 knockout screen for SubAB-induced cell death and identified various sialoglycan-related and membrane-trafficking genes. Analysis of glycan-deficient cells demonstrated that not only N-glycans but also O-glycans serve as SubAB receptors. In addition, SLC39A9, which is a predicted zinc transporter, as well as KDELRs and JTB, were required for SubAB to induce maximal cell death. Disruption of the SLC39A9 gene markedly reduced both complex-type N-glycans and core 1 O-glycans, and the O-glycan reduction was attributed to the reduction of core 1 synthase (C1GalT1). These results provide insights into the post-transcriptional regulation of glycosyltransferases by SLC39A9, as well as sialoglycan species as SubAB receptors.

The Synaptosome Associated Protein‐23 is Necessary for Skeletal Muscle Myogenesis

Alternative splicing is a post‐transcriptional RNA processing mechanism that occurs in more than 90% of human genes and allows for many proteins to arise from one gene. Membrane trafficking genes are regulated by alternative splicing during postnatal development1. The synaptosome associated protein‐23 (Snap23) is a member of the SNARE complex and is involved in the fusion of transport vesicles with the plasma membrane before cargo release2. We have shown that the Snap23 gene is regulated by alternative splicing. Exon 6, which contains 33 nucleotides, is skipped in fetal stages and is gradually included in adulthood. This transition is exclusive to striated muscles. SNAP23 is essential for mouse viability3; however, its role during muscle development is unclear. We hypothesize that SNAP23 is necessary for maintenance of muscle cell architecture and that alternative splicing is involved as a regulatory mechanism of muscle development.Murine C2C12 cell differentiation (myogenesis) replicates the Snap23 splicing transitions that we observed in muscle development. Thus, C2C12 cells are an appropriate model to investigate SNAP23 function and its regulation by alternative splicing. C2C12 cells were transfected with siRNAs to deplete SNAP23 protein expression in the undifferentiated stage and then differentiated for four days. Depletion of Snap23 led to a reduction in cell viability and fusion, indicating that Snap23 is required for myogenesis. Media collected from healthy differentiated cells (conditioned media) rescue the phenotype of Snap23 knock‐down cells. Immunofluorescence confirmed greater cell differentiation than those treated with standard differentiation media. This led us to hypothesize that SNAP23 mediates the secretion of a protein that is necessary for myogenesis. We coated plates with various concentrations of gelatin and observed that gelatin‐coated plates also restored cell differentiation. We are currently investigating fibronectin as a potential important protein, as it is a major component of gelatin and interacts with integrins on the plasma membrane.In parallel, to study the implications of alternative splicing on Snap23 pre‐mRNA, we used morpholino antisense oligonucleotides (MO) to redirect endogenous splicing decisions. Cells treated with Snap23‐MO expressed exclusively the short isoform throughout myogenesis while control cells express the long isoform in differentiated stages. In Snap23‐MO treated cells, we observed more myotubes with greater nuclei per myotube compared to the controls, suggesting that Snap23 splicing contributes to cell fusion and thus myogenesis.In conclusion, our data suggest that SNAP23 and its regulation by alternative splicing is involved in the fusion of myoblasts to form multinucleated myotubes. We propose that SNAP23 mediates the secretion of proteins necessary for myogenesis. Ongoing studies are directed to elucidate the molecular mechanisms behind the observed phenotypes.Support or Funding InformationNIH/NIGMS award (R25 GM089569) (to J. Gamarra UNC‐Chapel Hill PREP Scholar) and start up funds from UNC‐Chapel Hill (to J. Giudice)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A CRISPR Screen Identifies LAPTM4A and TM9SF Proteins as Glycolipid-Regulating Factors

SummaryGlycosphingolipids (GSLs) are produced by various GSL-synthesizing enzymes, but post-translational regulation of these enzymes is incompletely understood. To address this knowledge disparity, we focused on biosynthesis of globotriaosylceramide (Gb3), the Shiga toxin (STx) receptor, and performed a genome-wide CRISPR/CAS9 knockout screen in HeLa cells using STx1-mediated cytotoxicity. We identified various genes including sphingolipid-related genes and membrane-trafficking genes. In addition, we found two proteins, LAPTM4A and TM9SF2, for which physiological roles remain elusive. Disruption of either LAPTM4A or TM9SF2 genes reduced Gb3 biosynthesis, resulting in accumulation of its precursor, lactosylceramide. Loss of LAPTM4A decreased endogenous Gb3 synthase activity in a post-transcriptional mechanism, whereas loss of TM9SF2 did not affect Gb3 synthase activity but instead disrupted localization of Gb3 synthase. Furthermore, the Gb3-regulating activity of TM9SF2 was conserved in the TM9SF family. These results provide mechanistic insight into the post-translational regulation of the activity and localization of Gb3 synthase.

RNA interference screening identifies clathrin-B and cofilin-1 as mediators of MT1-MMP in endometrial cancer

The matrix metalloproteinases (MMPs) are implicated in tumor invasion and metastasis. Given their multiple tumor promoting roles, MMPs are promising targets for the treatment of metastatic cancer. Using a siRNA library screen of 140 membrane trafficking genes, we identified 41 genes in HEC-1B and 36 in Ishikawa cancer cells that decreased metalloproteinases activity. The 16 genes common in both cancer cell lines that decreased MMPs activity are involved in cargo sorting, vesicle formation and vesicle recycling. The top two genes clathrin-B and cofilin-1 were chosen for post hoc functional studies. Higher expression of both genes was confirmed in cancer cells and knockdown with respective siRNAs inhibited their invasive potential and matrix metalloproteinases activity. Membrane Type 1- Matrix Metalloproteinase (MT1-MMP) is a master switch proteinase and regulator of invasion and metastasis. A marked decrease in MT1-MMP expression and activity was seen in clathrin-B and cofilin-1 knockdown cancer cells which was associated with a marked decreased expression of invadopodia formation proteins. Our results suggest that the decreased expression of clathrin-B and cofilin-1 decreases the expression of MT1-MMP and results in attenuation of MT1-MMP at the cell surface, thus inhibiting tumor cell invasion and metastasis.

Identification and characterisation of a cryptic Golgi complex in Naegleria gruberi.

ABSTRACTAlthough the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here, we identify the Golgi complex in Naegleria, one of the first descriptions of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species of Naegleria and show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker (Ng)CopB by fluorescence in Naegleria gruberi, identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immunoelectron microscopy reveals that NgCOPB localises to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.This article has an associated First Person interview with the first author of the paper.

Caveolin-1-mediated Japanese encephalitis virus entry requires a two-step regulation of actin reorganization.

To investigate the detailed mechanism of Japanese encephalitis virus (JEV) cell entry. Utilize a siRNA library targeting cellular membrane trafficking genes to identify key molecules that mediate JEV entry into human neuronal cells. JEV enters human neuronal cells by caveolin-1-mediated endocytosis, which depends on a two-step regulation of actin cytoskeleton remodeling triggered by RhoA and Rac1: RhoA activation promoted the phosphorylation of caveolin-1, and then Rac1 activation facilitated caveolin-associated viral internalization. Specifically, virus attachment activates the EGFR-PI3K signaling pathway, thereby leading to RhoA activation. This work provides a detailed picture of the entry route and intricate cellular events following the entry of JEV into human neuronal cells, and promotes a better understanding of JEV entry.

Yeast as a simple eukaryotic model to study human diseases linked to membrane trafficking

Many human diseases are linked to mutations in genes encoding membrane trafficking proteins, among them the X-linked centronuclear myopathy (XLCNM) and ciliopathies. We use yeast Saccharomyces cerevisiae as a model system to study these human disease trafficking genes. It is a good model because yeast and human cells present similar intracellular organization and its membrane trafficking and metabolism are well characterized. We first studied the MTM1 gene, responsible for XLCNM, which codes for a myotubularin, a lipid phosphatase required for endosomal sorting. The heterologous expression of wild-type MTM1 or XLCNM patient mutants in yeast allowed comparing their in vivo phosphatase activity and their cellular function in trafficking. The yeast results showed that the phosphatase activity is not defective in patient mutants, further confirmed in the mouse Mtm1 KO model for myopathy [1]. Ciliopathies are complex genetic disorders that result from defects in the formation and/or function of the primary cilium. They include disorders such as Bardet-Biedl syndrome (BBS), Joubert syndrome (JBTS) and Alstrom syndrome (ALS) [2]. Cilia are involved in development and in signaling cascades. Despite, the identification of a large number of genes involved in ciliopathies (http://www.syscilia.org/goldstandard.shtml), the molecular and cellular mechanisms by which they cause the disease are largely unknown. In collaboration with the laboratory of Helene Dollfus, we aim at understanding the cellular roles of membrane trafficking genes involved in ciliopathies by using the yeast model system. Indeed, organization of the primary cilium depends on membrane trafficking since many genes linked to ciliopathies are involved in membrane trafficking [3].

An siRNA screen of membrane trafficking genes highlights pathways common to HIV-1 and M-PMV virus assembly and release.

The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.

Identification of cellular enhancing and restricting factors of dengue virus egress

Dengue has emerged as the most important life-threatening illness in the world, especially in Asian countries around Hong Kong, where the incidence of dengue hemorrhagic fever (DHF) is much greater than other continents. However, little is known about molecular and cellular processes sustaining egress of dengue virus in the host cell. To better understand the viral and cellular determinants of dengue virus egress, we have established a dengue VLP producing stable cell line (HeLa-prME) and demonstrated that dengue VLP was able to mimic the budding and egress process of dengue viruses so that it constitutes a safe and convenient tool for the study of egress of dengue virus. Under the support of RFCID grant, HeLa-prME cells were used to screen a siRNA library that included 122 cellular membrane trafficking genes. Our screen results revealed that knockdown of ADP-ribosylation factor (ARF) 1 and ARF6 had significant effects on VLP production by HeLa-prME cell. Experiments with other ARF proteins, which were not included in the siRNA library, showed that the ARF4/ARF5 double knockdown could inhibit VLP production but had no effect on secretion of other proteins such as soluble dengue E protein, suggesting the specificity of their involvement in VLP production. Further experiments using real virus showed that the depletion of ARF4/5 by siRNA could significantly reduced the replication of dengue 1 virus, dengue 4 virus and yellow fever virus, confirming the important role of ARF4 and ARF5 for not only dengue viruses but also other flaviviruses. Our study uncovered the importance of class II ARFs in the egress of dengue virus. Results from this project provided information on mechanism of dengue virus assembly and its dependence on cellular machineries.

Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

BackgroundDengue virus (DENV) is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined.ResultsIn this study, we have used a targeted small interfering RNA (siRNA) library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study.ConclusionsTogether, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti-viral strategies and treatment of DENV infection.

RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

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.

Potential roles for cellular cofactors in hepatitis C virus replication complex formation

Over 130 million people world-wide are chronically infected with hepatitis C virus (HCV). New antiviral treatment strategies are needed due to limitations with current therapy. The identification of cellular cofactors of infection has the potential to broadly expand our therapeutic targets. We recently reported an RNA interference screen of host membrane trafficking genes in HCV infection and replication and identified several cellular co-factors for viral replication. Phosphatidylinositol 4-kinase III alpha (PI4K-IIIα) was found to be essential for HCV replication. PI4K-IIIα co-localized with viral replication markers. Silencing of PI4K-IIIα by siRNAs prior to HCV infection prevented rearrangement of intracellular membranes associated with viral replication complexes, termed the membranous web. Our data suggest that PI4K-IIIα is involved in establishing HCV replication complexes, however the mechanism is unknown. From our analysis, along with several other studies that have identified cellular cofactors for HCV replication, we propose that PI4K-IIIα may nucleate replication complex formation by facilitating the interaction of viral and/or cellular proteins with cellular membrane-associated phospholipids.