Infection of Individual Tcells in vitro With HHV6, HIV, and HCV
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Slide 1 :
Syed Zaki SalahuddinCalifornia Institute of Molecular Medicine I have financial relationship(s) within the past 12 months relevant to my presentation with: California Institute of Molecular Medicine My presentation does not include discussion of off-label or investigational use.
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Infection of individual T-cells in vitro with HHV-6, HIV, and HCV Syed Zaki Salahuddin1, Dennis Revie2, Kathy Snyder2, Andre Godwin2, Renu Grewal1, John G. Prichard3, and Ann S. Kelley4 1California Institute of Molecular Medicine, Ventura, California, USA; 2California Lutheran University, Thousand Oaks, California, USA; 3Ventura County Medical Center, Ventura, California, USA; 4Ventura County Hematology-Oncology Specialists, Oxnard, California, USA. PP0055 APASL 2008. Seoul, Korea. March 25, 2008 APASL 2008. Seoul, Korea. March 25, 2008 APASL 2008. Seoul, Korea. March 25, 2008 APASL 2008. Seoul, Korea. March 25, 2008
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Abstract We have previously reported an in vitro system for isolating and replicating HCV from patients infected with this agent. Using this system, HCV was isolated from many infected patients. These isolates have been replicated in a variety of different cell types, hematopoietic and neuronal, for over four years. HCV isolated by this system show no significant differences from HCV found in the serum of patients. HIV-1 positive individuals are frequently also infected with HHV-6 and HCV. We isolate HIV-1 and HHV-6 from many of these patients for experimental purposes. We asked whether these three viruses could infect the same cells simultaneously. The host range for these viruses has previously been determined by us as well as others. All of them easily infect T-cells. Therefore, CEM (T-cells) were used for these experiments. We report here the successful infection of a CEM cell by all three viruses. PCR or RT-PCR analyses demonstrated that the CEM cells were productively infected by HHV-6, HCV, and HIV-1. Transmission electron microscopy (TEM) was used to show the co-infection of a single T-cell by all three viruses. The triply-infected cells were short lived, as these viruses are highly cytolytic. It was found, however, that HIV-1 and HCV co-infected cells lasted for several weeks. Viral replication in co-infected cells was unhindered, as 'dominance' was not observed in our experiments. HCV particles were present in the perinuclear space, suggesting their possible synthesis in the nucleus. This report is based on viruses produced in vitro in our laboratories.
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Introduction and Objectives Introduction HCV, HIV-1, and HHV-6 all can infect the same person. A culture system that can simultaneously replicate each of these viruses would enable their study. Objectives Develop an in vitro culture system to simultaneously culture HCV, HHV-6, and HIV-1. Use this system to study how these three viruses interact.
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Host range of these viruses HCV, HIV-1, and HHV-6 all infect T-cells. CEM T-cells were therefore used for the following experiments.
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Cell culture strategy All the transmissions by viruses used cell-free supernatants. The designations K1 through K7 don’t represent the order of the experiments.
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PCR and RT-PCR analysis of infected cell cultures A. HHV-6. The positive bands are 401 bp. Lane 1: 1 kb ladder Lane 2: Uninfected CEM cells Lane 3: K1 Lane 4: K2 Lane 5: K3 B. HHV-6. Lane 6 is K7. Different gel than gel A. C. HCV. The positive bands are 269 base pairs. Lane 1: Ladder. Lane 2: K5 Lane 4: K7 D. HCV. Lane 1: 1 kb ladder Lane 3: K6 E. HIV-1. The positive bands are 650 base pairs. Lane 1: 100 bp ladder Lane 2: Uninfected CEM cell supernatant Lane 3: K1 Lane 4: K2 Lane 5: K3 Lane 6: K4 F. HIV-1. Lane 1: 100 bp ladder Lane 2: K6 Lane 3: K7
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Infection of cells with viruses A. K6 cell infected with HHV-6 (arrows). The cell has typical features of an infected cell, including margination of the chromatin in the nucleus and the presence of many vacuoles in the cell. B. K2 cell showing extracellular HIV-1 particles (arrows). It is unclear whether the HIV-1 is outside the cell or in a vacuole inside the cell. C. K5 cell showing partial HCV particles inside a vacuole (arrow). D. K6 cell showing HCV particles adjacent to the cell (arrows). E. Inset showing HCV from Figure D.
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HCV and HIV-1 virions in a K7 cell TEM of HCV and HIV-1 extracellular particles adjacent to a K7 cell. Representative HIV-1 particles are indicated with black arrows, HCV with white arrows, and immature HIV-1 particles by a gray arrow. The HIV-1 particles are a little larger than HCV.
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Conclusions A single T-cell can be simultaneously infected by HCV, HIV-1, and HHV-6. These viruses replicate to a high titer in T-cells, as they can be seen in TEM. HCV virions can be found in the perinuclear space, suggesting their synthesis in the nucleus. This system will allow the study of interactions between these viruses.
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HCV, HIV-1, and HHV-6 in a single T-cell (CEM) TEM of triply-infected K7 cell. HIV-1 particle budding from the plasma membrane (arrow). Figure 6A is an example of HHV-6 budding from the nuclear membrane (black arrows) and HCV in the perinuclear space (white arrow).Figure 6B shows HCV particles in the cytoplasm. Figure 6C shows an HIV-1 particle outside the cell. Figure 6D shows HHV-6 (black arrows) and HCV (white arrow) in the perinuclear space.
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HCV, HIV-1, and HHV-6 in a single T-cell (CEM) TEM of triply-infected K7 cell. HIV-1 particle budding from the plasma membrane (arrow).
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HCV, HIV-1, and HHV-6 in a single T-cell (CEM) TEM of triply-infected K7 cell. Figure A is an example of HHV-6 budding from the nuclear membrane (black arrows) and HCV in the perinuclear space (white arrow).Figure B shows HCV particles in the cytoplasm. Figure C shows an HIV-1 particle outside the cell. Figure D shows HHV-6 (black arrows) and HCV (white arrow) in the perinuclear space.
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