Development of novel alphavirus expression and detection systems and characterization of oxidation's effect on viral replication

2014 Spring.Includes bibliographical references.Infectious diseases cause significant global suffering and death each year. Specifically, arthropod-borne viruses are emerging and re-emerging around the world and infecting millions of people. Mosquitoes that transmit these viruses are spreading to new regions of the world with naïve populations to serve as viral hosts. Climate change and human encroachment of habitat has brought mosquitoes into close proximity with humans. The viruses are evolving and are expanding their vector compatibility to include more than one species of mosquitoes. The combination of these elements results in a serious global need to develop ways to control or prevent arthropod-borne viruses. In order to discover novel antivirals and ways to inhibit these arboviruses, a better understanding of viral infection and replication is needed. This dissertation will describe a combination of projects that all aim to provide enhanced knowledge or tools to prevent, control, or treat arbovirus infection. Specifically, we improved the ability to express recombinant infectious alphaviruses, developed a novel system to detect alphavirus infection in mosquito cell culture and transgenic mosquitoes, and discovered a new role for oxidation during flavivirus replication. First, we successfully developed and established a method for transcribing infectious alphavirus RNA from a plasmid DNA platform. This approach provides an efficient way for producing high titer infectious recombinant alphavirus in multiple cell types that robustly express foreign proteins. Secondly, we optimized a system for detecting alphavirus infection in mosquito cells using the virus dependent subgenomic promoter to transcribe a reporter gene only during active infection. We demonstrated that mosquito cells can be stably transformed to transcribe an engineered viral reporter RNA that expresses a fluorescent reporter protein (mCherry) only in the presence of wild-type virus infection. The reporter protein is not detected in uninfected controls, but significant expression is readily detected during infection. Transgenic mosquitoes were also developed to transcribe the reporter RNA, which amplifies and expresses the reporter protein during infection. The transgenic mosquitoes are able to express a fluorescent reporter protein only during Sindbis virus (Alphavirus) infection, providing a novel mechanism to detect infection of wild-type virus in living mosquitoes. This transgenic reporter system is the first of its kind and demonstrated that a system based on our reporter RNAs could be optimized and used to specifically detect infected mosquitoes. Finally, I was able to study and characterize several aspects of viral RNA replication within the cell. Specifically, we identified that viral RNA replication is dependent on oxidative conditions. We determined individual residues from the flavivirus NS5 capping protein that are specifically involved in the oxidative enhancement of viral replication. Our work provided significant advances to the arbovirus field. We now have a novel method for producing recombinant alphaviruses that is more time, cost, and resource effective. We understand the ability of the subgenomic promoter to act as a virus inducible promoter to express foreign proteins only during infection to help detect or manipulate infection in mosquitoes. Finally, we have made significant discoveries on how RNA replication works on a molecular level within the cell and better understand the important role of oxidation on virus infection. The work and discoveries described in this dissertation have enhanced multiple aspects of arbovirus research and will hopefully strengthen our ability to fight and control arbovirus infections around the world

Oxidative stress influences positive strand RNA virus genome synthesis and capping

AbstractFlaviviruses are 5′ capped positive-stranded RNA viruses that replicate their genomes within endoplasmic reticulum-derived vesicles. Flaviviruses are well known to induce oxidative stress late in infection but it is unknown if oxidative stress plays a positive role in the viral RNA replication cycle. We therefore examined how oxidation affects flavivirus RNA replication. We found that antioxidant treatment reduced virus production, reduced the viral positive-to-negative strand RNA ratio, and resulted in the accumulation of uncapped positive-sense viral RNAs. Treatment of the NS5 RNA capping enzyme in vitro with oxidizing agents enhanced guanylyltransferase activity, indicating that the guanylyltransferase function of the flavivirus NS5 RNA capping enzyme is activated by oxidative conditions. Antioxidant treatment also reduced alphavirus RNA replication and protein expression while enhancing nsP1 capping activity. These findings suggest that RNA viruses may utilize oxidative stress induced during infection to help temporally control genome RNA capping and genome replication

Infectious alphavirus production from a simple plasmid transfection+

We have developed a new method for producing infectious double subgenomic alphaviruses from plasmids transfected into mammalian cells. A double subgenomic Sindbis virus (TE3'2J) was transcribed from a cytomegalovirus PolII promoter, which results in the production of infectious virus. Transfection of as little as 125 ng of plasmid is able to produce 1 × 108 plaque forming units/ml (PFU/ml) of infectious virus 48 hours post-transfection. This system represents a more efficient method for producing recombinant Sindbis viruses

Osteoarthritis and the rule of halves

<b>Background</b> Symptomatic osteoarthritis poses a major challenge to primary health care but no studies have related accessing primary care ("detection"), receiving recommended treatments ("treatment"), and achieving adequate control ("control").<p></p> <b>Objective</b> To provide estimates of detection, treatment, and control within a single population adapting the approach used to determine a Rule of Halves for other long-term conditions.<p></p> <b>Setting</b> General population.<p></p> <b>Participants</b> 400 adults aged 50+ years with prevalent symptomatic knee osteoarthritis.<p></p> <b>Design</b> Prospective cohort with baseline questionnaire, clinical assessment, and plain radiographs, and questionnaire follow-up at 18 and 36 months and linkage to primary care medical records.<p></p> <b>Outcome measures</b> "Detection" was defined as at least one musculoskeletal knee-related GP consultation between baseline and 36 months. "Treatment" was self-reported use of at least one recommended treatment or physiotherapy/hospital specialist referral for their knee problem at all three measurement points. Pain was "controlled" if characteristic pain intensity <5 out of 10 on at least two occasions.<p></p> <b>Results</b> In 221 cases (55.3%; 95%CI: 50.4, 60.1) there was evidence that the current problem had been detected in general practice. Of those detected, 164 (74.2% (68.4, 80.0)) were receiving one or more of the recommended treatments at all three measurement points. Of those detected and treated, 45 (27.4% (20.5, 34.3)) had symptoms under control on at least two occasions. Using narrower definitions resulted in substantially lower estimates.<p></p> <b>Conclusion</b> Osteoarthritis care does not conform to a Rule of Halves. Symptom control is low among those accessing health care and receiving treatment

Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase

The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%

Nonlinear Waves in Bose-Einstein Condensates: Physical Relevance and Mathematical Techniques

The aim of the present review is to introduce the reader to some of the physical notions and of the mathematical methods that are relevant to the study of nonlinear waves in Bose-Einstein Condensates (BECs). Upon introducing the general framework, we discuss the prototypical models that are relevant to this setting for different dimensions and different potentials confining the atoms. We analyze some of the model properties and explore their typical wave solutions (plane wave solutions, bright, dark, gap solitons, as well as vortices). We then offer a collection of mathematical methods that can be used to understand the existence, stability and dynamics of nonlinear waves in such BECs, either directly or starting from different types of limits (e.g., the linear or the nonlinear limit, or the discrete limit of the corresponding equation). Finally, we consider some special topics involving more recent developments, and experimental setups in which there is still considerable need for developing mathematical as well as computational tools.Comment: 69 pages, 10 figures, to appear in Nonlinearity, 2008. V2: new references added, fixed typo

School based working memory training: Preliminary finding of improvement in children’s mathematical performance

Working memory is a complex cognitive system responsible for the concurrent storage and processing of information. Ggiven that a complex cognitive task like mental arithmetic clearly places demands on working memory (e.g., in remembering partial results, monitoring progress through a multi-step calculation), there is surprisingly little research exploring the possibility of increasing young children’s working memory capacity through systematic school-based training. Tthis study reports the preliminary results of a working memory training programme, targeting executive processes such as inhibiting unwanted information, monitoring processes, and the concurrent storage and processing of information. Tthe findings suggest that children who received working memory training made significantly greater gains in the trained working memory task, and in a non-trained visual-spatial working memory task, than a matched control group. Moreover, the training group made significant improvements in their mathematical functioning as measured by the number of errors made in an addition task compared to the control group. Tthese findings, although preliminary, suggest that school-based measures to train working memory could have benefits in terms of improved performance in mathematics

Epigenetic Regulation of HIV-1 Latency by Cytosine Methylation

Human immunodeficiency virus type 1 (HIV-1) persists in a latent state within resting CD4+ T cells of infected persons treated with highly active antiretroviral therapy (HAART). This reservoir must be eliminated for the clearance of infection. Using a cDNA library screen, we have identified methyl-CpG binding domain protein 2 (MBD2) as a regulator of HIV-1 latency. Two CpG islands flank the HIV-1 transcription start site and are methylated in latently infected Jurkat cells and primary CD4+ T cells. MBD2 and histone deacetylase 2 (HDAC2) are found at one of these CpG islands during latency. Inhibition of cytosine methylation with 5-aza-2′deoxycytidine (aza-CdR) abrogates recruitment of MBD2 and HDAC2. Furthermore, aza-CdR potently synergizes with the NF-κB activators prostratin or TNF-α to reactivate latent HIV-1. These observations confirm that cytosine methylation and MBD2 are epigenetic regulators of HIV-1 latency. Clearance of HIV-1 from infected persons may be enhanced by inclusion of DNA methylation inhibitors, such as aza-CdR, and NF-κB activators into current antiviral therapies

The Secreted Metalloprotease ADAMTS20 Is Required for Melanoblast Survival

ADAMTS20 (A disintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kittm1Alf/+ and bt/bt;KitlSl/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases