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  1. HIV Infected Cells Have Depolarized Membrane Potentials and Increased Intracellular Calcium Levels [Download]

    Title: HIV Infected Cells Have Depolarized Membrane Potentials and Increased Intracellular Calcium Levels
    Author: Goydos, Ryan
    Description: Introduction/Background: Ion distribution between the extracellular, cytoplasmic, and organellar spaces creates membrane potentials which drive many of life’s processes. This bioelectric membrane potential, driven by ion channel and pump activity, can be harnessed to allow or prevent entry of signaling mediators like Ca2+ into the cytoplasm. Several HIV proteins (Vpu, Env, Vpr, and Nef) have been reported to function as ion channels or alter ion channel activity. This activity likely influences cell fate including activation and apoptosis. Hypothesis: HIV depolarizes the plasma membrane and alters intracellular calcium levels. Changing the polarization of the plasma membrane would alter the levels of HIV infection. Methods: HIV infected cells were identified using a broadly neutralizing anti-Env antibody (PG9) conjugated to AlexaFluor-647. Membrane potential measurements were done by flow cytometry using the DiBAC4(3) dye as previously reported. Intracellular Ca2+ measurements were also done by flow cytometry using the Fluo-4 dye. Ionomycin and PMA were used to show the contrast in intracellular Ca2+ levels between infected and uninfected cells. To assess the effects of membrane potential changes on HIV replication, 200μM diazoxide was added to cells during infections. Results: HIV infected cells consistently had depolarized membrane potentials in both primary cells and cell lines. When cells were cultured with a depolarizing agent, diazoxide, there was an increase in HIV-infected cells. This membrane depolarization was accompanied by an increased resting level of intracellular Ca2+ in infected cells. Following addition of ionomycin, there was a drastic difference in Ca2+ flow between uninfected and HIV-infected cells. In uninfected cells, the addition of ionomycin induced an influx of Ca2+ while PMA had little effect. In contrast, both ionomycin and PMA induced a large efflux of Ca2+ from HIV infected cells.
    Keywords: Research Days 2018, HIV, Microbiology, Immunology, Tropical medicine
    Date Uploaded: 04/21/2018
  2. Differential Gene Expression in Primary Human CD4+ T Cells Induced by HIV Nef [Download]

    Title: Differential Gene Expression in Primary Human CD4+ T Cells Induced by HIV Nef
    Author: Goydos, Ryan
    Description: The Nef protein is responsible for T-cell activation and formation of a persistent state of infection in HIV disease. Findings from the Sydney Blood Bank Cohort and other case studies indicate that individuals infected with a variant of HIV containing a defective nef gene displayed a slower development to AIDS compared to individuals infected with wild-type HIV. Our study investigates the role of the Nef protein on cellular gene transcription in infected primary CD4+ T cells with the goal of understanding how HIV Nef promotes viral replication and subsequent cellular pathology.
    Keywords: Research Days 2017, HIV, RNASeq, Transcriptome, AIDS
    Date Uploaded: 04/17/2017