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In JoVE (1)
Other Publications (6)
Articles by Rachel Kaletsky in JoVE
JoVE Neuroscience
C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays
1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Lewis-Sigler Institute for Integrative Genomics, Princeton University
Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.
Other articles by Rachel Kaletsky on PubMed
HIV-1 Integrates into Resting CD4+ T Cells Even at Low Inoculums As Demonstrated with an Improved Assay for HIV-1 Integration
Human Immunodeficiency Virus Type 1 (HIV-1) establishes a latent reservoir early in infection that is resistant to the host immune response and treatment with highly active antiretroviral therapy (HAART). The best understood of these reservoirs forms in resting CD4(+) T cells. While it remains unclear how reservoirs form, a popular model holds that the virus can only integrate in activated CD4(+) T cells. Contrary to this model, our previous results suggest that HIV-1 can integrate directly into the genomes of resting CD4(+) T cells. However, a limitation of our previous studies was that they were conducted at high viral inoculum and these conditions may lead to cellular activation or saturation of restriction factors. In the present study, we tested if our previous findings were an artifact of high inoculum. To do this, we enhanced the sensitivity of our integration assay by incorporating a repetitive sampling technique that allowed us to capture rare integration events that occur near an Alu repeat. The new technique represents a significant advance as it enabled us to measure integration accurately down to 1 provirus/well in 15,000 genomes--a 40-fold enhancement over our prior assay. Using this assay, we demonstrate that HIV can integrate into resting CD4(+) T cells in vitro even at low viral inoculum. These findings suggest there is no threshold number of virions required for HIV to integrate into resting CD4(+) T cells.
Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity
Cellular cathepsins are required for Ebola virus infection and are believed to proteolytically process the Ebola virus glycoprotein (GP) during entry. However, the significance of cathepsin cleavage during infection remains unclear. Here we demonstrate a role for cathepsin L (CatL) cleavage of Ebola virus GP in the generation of a stable 18-kDa GP1 viral intermediate that exhibits increased binding to and infectivity for susceptible cell targets. Cell binding to a lymphocyte line was increased when CatL-proteolysed pseudovirions were used, but lymphocytes remained resistant to Ebola virus GP-mediated infection. Genetic removal of the highly glycosylated mucin domain in Ebola virus GP resulted in cell binding similar to that observed with CatL-treated full-length GP, and no overall enhancement of binding or infectivity was observed when mucin-deleted virions were treated with CatL. These results suggest that cathepsin cleavage of Ebola virus GP facilitates an interaction with a cellular receptor(s) and that removal of the mucin domain may facilitate receptor binding. The influence of CatL in Ebola virus GP receptor binding should be useful in future studies characterizing the mechanism of Ebola virus entry.
An Initial Characterization of the Mercury Resistance (mer) System of the Thermophilic Bacterium Thermus Thermophilus HB27
The evolutionary origin of the broadly distributed mer system, which plays an important role in mercury detoxification and biogeochemistry, is presently unknown. The phylum Deinococcus/Thermus was found to be one of the deepest-branching bacterial lineage to have a homolog of merA, which specifies reduction of ionic to elemental mercury, and the mercuric reductase (MerA) of Thermus thermophilus HB27 was found to be basal to all bacterial MerA when this protein's phylogeny was constructed. A merA mutant of HB27 was fourfolds more sensitive to mercury toxicity than the wild type (wt), and lost detectable MerA-specific activities. The merA gene in HB27 was transcribed on a polycistronic message downstream from ORF encoding for homologs of O-acetyl-l-homoserine/O-acetyl-serine (OAH/OAS) sulfhydrylase and MerR, the mer operon transcription regulator, from a promoter located 69 nucleotides upstream of the sulfhydrylase translation start codon. The transcription of the putative mer operon in HB27 was induced 66.8+/-15.8-fold by exposure to 1 muM HgCl2. The optimal temperature for MerA-specific activity corresponded to this strain's optimal growth temperature, 70 degrees C. Thus, T. thermophilus is the earliest mercury-resistant bacterium identified to date, a finding consistent with the hypothesis that the mer system originated among thermophilic microorganisms from geothermal environments.
Tetherin-mediated Restriction of Filovirus Budding is Antagonized by the Ebola Glycoprotein
Mammalian cells employ numerous innate cellular mechanisms to inhibit viral replication and spread. Tetherin, also known as Bst-2 or CD317, is a recently identified, IFN-induced, cellular response factor that blocks release of HIV-1 and other retroviruses from infected cells. The means by which tetherin retains retroviruses on the cell surface, as well as the mechanism used by the HIV-1 accessory protein Vpu to antagonize tetherin function and promote HIV-1 release, are unknown. Here, we document that tetherin functions as a broadly acting antiviral factor by demonstrating that both human and murine tetherin potently inhibit the release of the filovirus, Ebola, from the surface of cells. Expression of the Ebola glycoprotein (GP) antagonized the antiviral effect of human and murine tetherin and facilitated budding of Ebola particles, as did the HIV-1 Vpu protein. Conversely, Ebola GP could substitute for Vpu to promote HIV-1 virion release from tetherin-expressing cells, demonstrating a common cellular target for these divergent viral proteins. Ebola GP efficiently coimmunoprecipitated with tetherin, suggesting that the viral glycoprotein directly interferes with this host antiviral factor. These results demonstrate that tetherin is a cellular antiviral factor that restricts budding of structurally diverse enveloped viruses. Additionally, Ebola has evolved a highly effective strategy to combat this antiviral response elicited in the host during infection.
The Role of Insulin/IGF-like Signaling in C. Elegans Longevity and Aging
Aging is characterized by general physiological decline over time. A hallmark of human senescence is the onset of various age-related afflictions including neurodegeneration, cardiovascular disease and cancer. Although environmental and stochastic factors undoubtedly contribute to the increased incidence of disease with age, recent studies suggest that intrinsic genetic determinants govern both life span and overall health. Current aging research aims at achieving the 'longevity dividend', in which life span extension in humans is accomplished with a concomitant increase in the quality of life (Olshansky et al., 2007). Significant progress has been made using model organisms, especially the nematode worm Caenorhabditis elegans, to delineate the genetic and biochemical pathways involved in aging to identify strategies for therapeutic intervention in humans. In this review, we discuss how C. elegans has contributed to our understanding of insulin signaling and aging.
A Small-molecule Oxocarbazate Inhibitor of Human Cathepsin L Blocks Severe Acute Respiratory Syndrome and Ebola Pseudotype Virus Infection into Human Embryonic Kidney 293T Cells
A tetrahydroquinoline oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human cathepsin L (EC 3.4.22.15) and as an entry blocker of severe acute respiratory syndrome (SARS) coronavirus and Ebola pseudotype virus. In the cathepsin L inhibition assay, the oxocarbazate caused a time-dependent 17-fold drop in IC(50) from 6.9 nM (no preincubation) to 0.4 nM (4-h preincubation). Slowly reversible inhibition was demonstrated in a dilution assay. A transient kinetic analysis using a single-step competitive inhibition model provided rate constants of k(on) = 153,000 M(-1)s(-1) and k(off) = 4.40 x 10(-5) s(-1) (K(i) = 0.29 nM). The compound also displayed cathepsin L/B selectivity of >700-fold and was nontoxic to human aortic endothelial cells at 100 muM. The oxocarbazate and a related thiocarbazate (PubChem CID 16725315) were tested in a SARS coronavirus (CoV) and Ebola virus-pseudotype infection assay with the oxocarbazate but not the thiocarbazate, demonstrating activity in blocking both SARS-CoV (IC(50) = 273 +/- 49 nM) and Ebola virus (IC(50) = 193 +/- 39 nM) entry into human embryonic kidney 293T cells. To trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity-based probe biotin-Lys-C5 alkyl linker-Tyr-Leu-epoxide (DCG-04) was used to label the active site of cysteine proteases in 293T lysates. The reduction in active cathepsin L in inhibitor-treated cells correlated well with the observed potency of inhibitors observed in the virus pseudotype infection assay. Overall, the oxocarbazate CID 23631927 was a subnanomolar, slow-binding, reversible inhibitor of human cathepsin L that blocked SARS-CoV and Ebola pseudotype virus entry in human cells.
