SCIENTIFIC CORES
Bacterial Protein Expression Core
Biochemistry Core Cell Biology and Imaging Core Computation Core EM Crystallography Core EM Tomography Core Eukaryotic Protein Expression Core Fluorescence Spectroscopy Core Protein Interactions Core Protein NMR Spectroscopy Core RNA Structure and Dynamics Core Tissue EM Core Virology Core Virus Imaging Core X-ray Crystallography Core |
Virology Core
Director: Wes Sundquist, PhD Manager: TBN The Virology Core conducts research in molecular virology and expands the capabilities of Center members by providing access to methods for producing HIV, analyzing viral replication, and siRNA technology and other approaches for validating and characterizing candidate host factors (as well as more specialized assays). Resources and Capabilities Facilities Tissue culture and molecular virology are performed in two laboratories at the University of Utah: 1) a fully equipped BSL-3 laboratory that is certified for work with infectious HIV (499 sq. ft., two tissue culture hoods), and 2) a conventional tissue culture laboratory that follows BSL-2+ safety procedures (three hoods, 350 sq. ft.). Both laboratories are equipped for tissue culture and virus growth, with hoods, refrigerators, chilled centrifuges and an ultracentrifuge, incubators, liquid nitrogen freezers, microscopes, etc. The BSL-2+ lab is used for routine tissue culture applications, viral vector work, and protein expression in human and insect cells. The BSL-3 lab is used exclusively for growth and research of HIV-1 and other potentially pathogenic retroviruses. Safety features of the BSL-3 lab include: a combination exterior lock, a negative air pressure system with a continuously monitored alarm, an internal autoclave system, an airlock antechamber room used for lab storage and for gowning, external liquid nitrogen and CO2 systems, and auto-timed, laboratory-wide UV sterilization lights. All experiments are performed by fully trained personnel under BSL-3 safety conditions. Viruses, Vectors, and Cells The Core can produce and analyze a number of infectious HIV-1 strains with different tropisms (NL4-3, HXB2, BRU2, YU-2, R7-BaL and the O-type MVP5180 virus). Available retroviral vectors include: pCMVΔR8.9, pCMVΔR8.2, and pCMV-gag-pol-RRE, with packagable reporters that express GFP and luciferase. Virus/vector production is typically initiated by transient transfection of 293T cells with full-length proviral clones (e.g., pR9). Tissue culture cell lines (typically 293T, CD4+ HeLa, SupT1, CEM, Jurkat, or K562) are usually used to allow rapid and reproducible analyses of high titer HIV-1 infections, but key observations can be confirmed in primary CD4+ T cells or monocyte derived macrophages. Monitoring Viral Replication and Protein Production Viral titers are typically measured calorimetrically in single-cycle “MAGIC” assays using the P4 HeLa.CD4.LTR-β-gal cell line. HIV-1 growth curves can also be performed in T cells or HeLa-CD4 cells, with levels of virus in cell-free supernatants analyzed by: 1) p24 ELISA, 2) RT, or 3) qRT-PCR assays. The requested fluorescence microscope will enable us to measure viral titers within the BSL-3 laboratory using Rev- or Tat-inducible GFP reporter cells (Rev-CEM, CEM-GFP). Viral protein levels and processing are detected by western blot analyses using various HIV-specific or other antisera, following concentration of virions by high speed centrifugation through sucrose cushions, and/or following: 1) subtilisin treatment to remove contaminating vesicles, or 2) sucrose or Optiprep gradients. Viral reverse transcription and nuclear transport can also be analyzed by real-time qPCR assays for early (R/U5) or late (R/5NC) reverse transcripts or 2LTR circles (R/R2). RNA Interference In 2001, we were the first to employ siRNA to test the effects of host protein depletion on viral replication and to use siRNA-resistant constructs to “rescue” these depletion phenotypes. These experiments constitute a “genetic” system for mammalian tissue culture that enables us to study the role of host proteins on the HIV-1 life cycle. The Virology core also has access to the University of Utah Amaxa Nucleofector Core Facility for DNA and RNA transfection of difficult cell lines and primary cells. shRNAs are typically delivered using a custom lentiviral vector engineered to co-express an shRNA, a sortable marker (DsRed), and an siRNA-resistant construct. Expression levels of siRNA-resistant constructs can be “tuned” to optimize rescue levels using a family of vectors containing different CMV promoters. Large Scale Viral Preparations To prepare virus on a large scale, proviral plasmid DNA is transfected into 293T cells (calcium phosphate) and the released virions are concentrated by ultracentrifugation. Concentrated virions are purified on 20-60% sucrose gradients, assayed by p24 ELISA or western blotting, and resuspended in small volumes for EM analyses. This procedure typically yields >100 ng CA protein. Analogous approaches are used to purify membrane-stripped viral cores for structural, biochemical and functional studies.  |