Hameeda Sultana

RNA interference screen for human genes associated with West Nile virus infection.

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus–host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV–human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold.

In the United States, Ixodes scapularis ticks overwinter in the Northeast and Upper Midwest and transmit the agent of human granulocytic anaplasmosis, Anaplasma phagocytophilum, among other pathogens. We now show that the presence of A. phagocytophilum in I. scapularis ticks increases their ability to survive in the cold. We identified an I. scapularis antifreeze glycoprotein, designated IAFGP, and demonstrated via RNAi knockdown studies the importance of IAFGP for the survival of I. scapularis ticks in a cold environment. Transfection studies also show that IAFGP increased the viability of yeast cells subjected to cold temperature. Remarkably, A. phagocytophilum induced the expression of iafgp, thereby increasing the cold tolerance and survival of I. scapularis. These data define a molecular basis for symbiosis between a human pathogenic bacterium and its arthropod vector and delineate what we believe to be a new pathway that may be targeted to alter the life cycle of this microbe and its invertebrate host.

Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I

Caspase-12 has been shown to negatively modulate inflammasome signaling during bacterial infection. Its function in viral immunity, however, has not been characterized. We now report an important role for caspase-12 in controlling viral infection via the pattern-recognition receptor RIG-I. After challenge with West Nile virus (WNV), caspase-12-deficient mice had greater mortality, higher viral burden and defective type I interferon response compared with those of challenged wild-type mice. In vitro studies of primary neurons and mouse embryonic fibroblasts showed that caspase-12 positively modulated the production of type I interferon by regulating E3 ubiquitin ligase TRIM25–mediated ubiquitination of RIG-I, a critical signaling event for the type I interferon response to WNV and other important viral pathogens.

Fusion Loop Peptide of the West Nile Virus Envelope Protein Is Essential for Pathogenesis and Is Recognized by a Therapeutic Cross-Reactive Human Monoclonal Antibody

West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.

Cyclase-Associated Protein is Essential for the Functioning of the Endo-Lysosomal System and Provides a Link to the Actin Cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase‐associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase‐associated protein associates and interacts with subunits of the highly conserved vacuolar H+‐ATPase (V‐ATPase) and co‐localizes to some extent with the V‐ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo‐lysosomal system, as distribution and morphology of V‐ATPase‐ and Nramp1‐decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V‐ATPase, had a more severe effect on CAP bsr than on wild‐type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein‐CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V‐ATPase in CAP bsr was drastically altered upon hypo‐osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper‐osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo‐lysosomal system in Dictyostelium.

Semaphorin 7A Contributes to West Nile Virus Pathogenesis through TGF-β1/Smad6 Signaling

Semaphorin 7A (Sema7A) is a membrane-associated/secreted protein that plays an essential role in connecting the vertebrate neuronal and immune systems. However, the role of Sema7A has not been elucidated in viral pathogenesis. In this study, we show that abrogation of Sema7A protects mice from lethal West Nile virus (WNV) infection. Mice lacking Sema7A showed increased survival, reduced viral burden, and less blood–brain barrier permeability upon WNV infection. Increased Sema7A levels were evident in murine tissues, as well as in murine cortical neurons and primary human macrophages upon WNV infection. Treatment with Sema7A Ab blocked WNV infection in both of these cell types. Furthermore, Sema7A positively regulates the production of TGF-β1 and Smad6 to facilitate WNV pathogenesis in mice. Collectively, these data elucidate the role of Sema7A in shared signaling pathways used by the immune and nervous systems during viral pathogenesis that may lead to the development of Sema7A-blocking therapies for WNV and possibly other flaviviral infections.

The Use of Metagenomic Approaches to Analyze Changes in Microbial Communities

Microbes are the most abundant biological entities found in the biosphere. Identification and measurement of microorganisms (including viruses, bacteria, archaea, fungi, and protists) in the biosphere cannot be readily achieved due to limitations in culturing methods. A non-culture based approach, called “metagenomics”, was developed that enabled researchers to comprehensively analyse microbial communities in different ecosystems. In this study, we highlight recent advances in the field of metagenomics for analyzing microbial communities in different ecosystems ranging from oceans to the human microbiome. Developments in several bioinformatics approaches are also discussed in context of microbial metagenomics that include taxonomic systems, sequence databases, and sequence-alignment tools. In summary, we provide a snapshot for the recent advances in metagenomics approach for analyzing changes in the microbial communities in different ecosystems.

Antivirulence properties of an antifreeze protein.

As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

Ticks elicit variable fibrinogenolytic activities upon feeding on hosts with different immune backgrounds

Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Furthermore, the LC-MS/MS and quantitative real-time-PCR analysis followed by inhibitor and antibody-blocking assays revealed that the arthropod HSP70-like molecule contributes to differential fibrinogenolysis during tick feeding. Collectively, these results not only indicate that ticks elicit variable fibrinogenolysis upon feeding on hosts with different immune backgrounds but also provide insights for the novel role of arthropod HSP70-like molecule in fibrinogenolysis during blood feeding.

Expression of Ixodes scapularis Antifreeze Glycoprotein Enhances Cold Tolerance in Drosophila melanogaster

Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies.