[Show abstract][Hide abstract] ABSTRACT: Cells contain several inositol pyrophosphates (PP-InsPs; also known as diphosphoinositol polyphosphates), which play pivotal roles in cellular and organismic homeostasis. It has been proposed that determining mechanisms of compartmentation of the synthesis of a particular PP-InsP is key to understanding how each of them may exert a specific function. Human PPIP5K2 (hPPIP5K2), one of the key enzymes that synthesizes PP-InsPs, contains a putative consensus sequence for a nuclear localization signal (NLS). However, such in silico analysis has limited predictive power, and may be complicated by phosphorylation events that can dynamically modulate NLS function. We investigated if this candidate NLS is functional and regulated, using the techniques of cell biology, mutagenesis and mass spectrometry.
Multiple sequence alignments revealed that the metazoan PPIP5K2 family contains a candidate NLS within a strikingly well-conserved 63 amino-acid domain. By analyzing the distribution of hPPIP5K2-GFP in HEK293T cells with the techniques of confocal microscopy and imaging flow cytometry, we found that a distinct pool of hPPIP5K2 is present in the nucleus. Imaging flow cytometry yielded particular insight into the characteristics of the nuclear hPPIP5K2 sub-pool, through a high-throughput, statistically-robust analysis of many hundreds of cells. Mutagenic disruption of the candidate NLS in hPPIP5K2 reduced its degree of nuclear localization. Proximal to the NLS is a Ser residue (S1006) that mass spectrometry data indicate is phosphorylated inside cells. The degree of nuclear localization of hPPIP5K2 was increased when S1006 was rendered non-phosphorylatable by its mutation to Ala. Conversely, a S1006D phosphomimetic mutant of hPPIP5K2 exhibited a lower degree of nuclear localization.
The current study describes for the first time the functional significance of an NLS in the conserved PPIP5K2 family. We have further demonstrated that there is phosphorylation of a Ser residue that is proximal to the NLS of hPPIP5K2. These conclusions draw attention to nuclear compartmentation of PPIP5K2 as being a physiologically relevant and covalently-regulated event. Our study also increases general insight into the consensus sequences of other NLSs, the functions of which might be similarly regulated.
[Show abstract][Hide abstract] ABSTRACT: The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.
[Show abstract][Hide abstract] ABSTRACT: West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans.
[Show abstract][Hide abstract] ABSTRACT: West Nile (WNV), dengue (DENV) and yellow fever (YFV) viruses are (re)emerging, mosquito-borne flaviviruses that cause human disease and mortality worldwide. Alterations in mosquito gene expression common and unique to individual flaviviral infections are poorly understood. Here, we present a microarray analysis of the Aedes aegypti transcriptome over time during infection with DENV, WNV or YFV. We identified 203 mosquito genes that were ≥ 5-fold differentially up-regulated (DUR) and 202 genes that were ≥ 10-fold differentially down-regulated (DDR) during infection with one of the three flaviviruses. Comparative analysis revealed that the expression profile of 20 DUR genes and 15 DDR genes was quite similar between the three flaviviruses on D1 of infection, indicating a potentially conserved transcriptomic signature of flaviviral infection. Bioinformatics analysis revealed changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. We also demonstrate that virally-regulated gene expression is tissue-specific. The overexpression of several virally down-regulated genes decreased WNV infection in mosquito cells and Aedes aegypti mosquitoes. Among these, a pupal cuticle protein was shown to bind WNV envelope protein, leading to inhibition of infection in vitro and the prevention of lethal WNV encephalitis in mice. This work provides an extensive list of targets for controlling flaviviral infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.
[Show abstract][Hide abstract] ABSTRACT: West Nile and dengue viruses are (re)emerging mosquito-borne flaviviruses that cause significant morbidity and mortality in man. The identification of mosquito proteins that associate with flaviviruses may provide novel targets to inhibit infection of the vector or block transmission to humans. Here, a tandem affinity purification (TAP) assay was used to identify 18 mosquito proteins that interact with dengue and West Nile capsid, envelope, NS2A or NS2B proteins. We further analyzed the interaction of mosquito cadherin with dengue and West Nile virus envelope protein using co-immunoprecipitation and immunofluorescence. Blocking the function of select mosquito factors, including actin, myosin, PI3-kinase and myosin light chain kinase, reduced both dengue and West Nile virus infection in mosquito cells. We show that the TAP method may be used in insect cells to accurately identify flaviviral-host protein interactions. Our data also provides several targets for interrupting flavivirus infection in mosquito vectors.
[Show abstract][Hide abstract] ABSTRACT: West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.
[Show abstract][Hide abstract] ABSTRACT: Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses antiviral restriction factors to defend against infection. To find host cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing. We discovered that the interferon-inducible transmembrane proteins IFITM1, 2, and 3 restrict an early step in influenza A viral replication. The IFITM proteins confer basal resistance to influenza A virus but are also inducible by interferons type I and II and are critical for interferon's virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus and West Nile virus. Collectively this work identifies a family of antiviral restriction factors that mediate cellular innate immunity to at least three major human pathogens.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Immunology 08/2009; 183(1):650-60. DOI:10.4049/jimmunol.0900093 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: West Nile virus (WNV) is an emerging human pathogen for which specific antiviral therapy has not been developed. The therapeutic potential of RNA interference (RNAi) as a sequence-specific inhibitor of WNV has been well demonstrated. Although multiple siRNA targets have been identified within the genomic coding region, targets within the untranslated regions (UTR), which encode cis-acting regulatory elements, remain relatively unknown. In WNV and other flaviviruses, UTRs are located at the genomic termini. These regions form complex secondary structures, which pose difficulty when designing effective siRNA targets. In this study, we report the identification of siRNA targets in the WNV 3' UTR. These targets were selected by siRNA predictor algorithms, and synthesized as short hairpin RNA sequences from a plasmid-based expression system. Vero cells stably expressing these sequences had greatly diminished ability to support WNV replication but not the related dengue virus, demonstrating that the siRNAs were effective and suppressed WNV viral replication in a sequence-specific manner. The siRNAs developed in this study could function as potential antiviral therapeutics and as genetic tools to investigate the role of 3' UTR in WNV pathogenesis.
Antiviral research 02/2009; 82(3):166-8. DOI:10.1016/j.antiviral.2008.12.007 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms of cellular entry of dengue and West Nile viruses are not well characterized. We show that both these viruses enter HeLa cells by clathrin-dependent endocytosis and require vacuolar acidic pH. Inhibition of the GTPase Rab 5 or 7, which regulates transport to early or late endosomes, respectively, demonstrated that Rab 5 was essential for survival of both dengue and West Nile virus. These data broaden our understanding of the pathways required for productive dengue and West Nile virus infection and may facilitate new strategies for combating disease.
Journal of Virology 06/2007; 81(9):4881-5. DOI:10.1128/JVI.02210-06 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference-mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum-infected mice. A. phagocytophilum migrated normally from A. phagocytophilum-infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.
Journal of Experimental Medicine 07/2006; 203(6):1507-17. DOI:10.1084/jem.20060208 · 13.91 Impact Factor